Penicillin-g acylases

ABSTRACT

The present disclosure relates to engineered penicillin G acylase (PGA) enzymes having improved properties, polynucleotides encoding such enzymes, compositions including the enzymes, and methods of using the enzymes.

The present application claims priority to U.S. Prov. Pat. Appln. Ser.No. 62/158,118, filed May 7, 2015, hereby incorporated by reference inits entirety for all purposes.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing concurrently submitted herewith under 37 C.F.R.§1.821 in a computer readable form (CRF) via EFS-Web as file nameCX2-149US1_ST25.txt is herein incorporated by reference. The electroniccopy of the Sequence Listing was created on Apr. 28, 2016, with a filesize of 88 Kbytes.

FIELD OF THE INVENTION

The present disclosure relates to engineered penicillin G acylase (PGA)enzymes, polynucleotides encoding the enzymes, compositions comprisingthe enzymes, and methods of using the engineered PGA enzymes.

BACKGROUND OF THE INVENTION

Penicillin G acylase (PGA) (penicillin amidase, EC 3.5.1.11) catalyzesthe cleavage of the amide bond of penicillin G (benzylpenicillin) sidechain. The enzyme is used commercially in the manufacture of6-amino-penicillanic acid (6-APA) and phenyl-acetic acid (PAA). 6-APA isa key compound in the industrial production of semi-synthetic β-lactamantibiotics such as amoxicillin, ampicillin and cephalexin. Thenaturally occurring PGA enzyme shows instability in commercialprocesses, requiring immobilization on solid substrates for commercialapplications. PGA has been covalently bonded to various supports and PGAimmobilized systems have been reported as useful tools for the synthesisof pure optical isomers. Attachment to solid surfaces, however, leads tocompromised enzyme properties, such as reduced activity and/orselectivity, and limitations to solute access. Moreover, althoughattachment to solid substrates allows capture of enzymes and reuse inadditional processing cycles, the stability of the enzyme is such thatsuch applications may be limited. The enzymatic catalysis by PGA ofpenicillin G to 6-APA is regiospecific (it does not cleave the lactamamide bond) and stereospecific. The production of 6-APA constitutesperhaps the largest utilization of enzymatic catalysis in the productionof pharmaceuticals. The enzymatic activity of PGA, associated with thephenacetyl moiety, allows the stereospecific hydrolysis of a richvariety of phenacetyl derivatives of primary amines as well as alcohols.

SUMMARY OF THE INVENTION

The present disclosure relates to engineered penicillin G acylase (PGA)enzymes, polynucleotides encoding the enzymes, compositions comprisingthe enzymes, and methods of using the engineered PGA enzymes.

The present invention provides engineered penicillin G acylases capableof removing the A1/B1/B29 tri-phenyl acetate protecting groups frominsulin to produce free insulin, wherein the penicillin G acylase is atleast about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or more identical to SEQ ID NO:2, 4, 6, 8,10, and/or 12. In some embodiments, the present invention providesengineered penicillin G acylases capable of removing the A1/B1/B29tri-phenyl acetate protecting groups from insulin to produce freeinsulin, wherein the penicillin G acylase is at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moreidentical to SEQ ID NO:2, 4, 6, 8, 10, and/or 12. In some additionalembodiments, the present invention provides engineered penicillin Gacylases capable of removing the A1/B1/B29 tri-phenyl acetate protectinggroups from insulin to produce free insulin, wherein the penicillin Gacylase comprises SEQ ID NO:2, 4, 6, 8, 10, and/or 12. In some furtherembodiments, the penicillin G acylase comprises at least one mutation asprovided in Table 5.1, Table 6.2, and/or Table 6.3.

The present invention also provides a penicillin G acylase encoded by apolynucleotide sequence having at least about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or moresequence identity to a sequence selected from SEQ ID NOS:3, 5, 7, 9, and11.

In some embodiments, the penicillin G acylase encoded by apolynucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to asequence selected from SEQ ID NOS:3, 5, 7, 9, and 11. In someembodiments, the penicillin G acylase encoded by a polynucleotidesequence selected from SEQ ID NOS:3, 5, 7, 9, and 11. The presentinvention also provides vectors comprising the polynucleotide sequencesprovided herein (e.g., SEQ ID NOS:3, 5, 7, 9, and/or 11). The presentinvention also provides host cells comprising the vectors providedherein (e.g., vectors comprising the polynucleotide sequences of SEQ IDNOS:3, 5, 7, 9, and/or 11).

The present invention also provides methods for producing free insulin,comprising: i) providing at least one engineered penicillin G acylaseprovided herein, and insulin comprising A1/B1/B29 tri-phenyl acetateprotecting groups; and ii) exposing the engineered penicillin G acylaseto the insulin comprising A1/B1/B29 tri-phenyl acetate protectinggroups, under conditions such that the engineered penicillin G acylaseremoves the A1/B1/B29 tri-phenyl acetate protecting groups and freeinsulin is produced. In some embodiments of the methods, the penicillinG acylase is at least about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, or more identical to SEQ IDNO:2, 4, 6, 8, 10, and/or 12. In some embodiments of the methods, thepenicillin G acylase is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO:2, 4,6, 8, 10, and/or 12. In some further embodiments of the methods,penicillin G acylase comprises SEQ ID NO:2, 4, 6, 8, 10, and/or 12. Insome embodiments, the engineered penicillin G acylase produces more than90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more free insulin.The present invention also provides compositions comprising free insulinproduced according to the method(s) provided herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a graph showing the substrate inhibition activityobserved with Variant 1.

FIG. 2 provides a graph showing the amount of free insulin producedusing seven variant PGAs.

FIG. 3 provides a graph showing % yield of free insulin produced usingthree variant PGAs.

FIG. 4 provides a graph showing % yield of free insulin produced usingthree variant PGAs in the presence of DMSO in the reactions.

DESCRIPTION OF THE INVENTION

The present invention provides engineered penicillin G acylases (PGA)that are capable of cleaving penicillin to phenylacetic acid and6-aminopenicillanic acid (6-APA), which is a key intermediate in thesynthesis of a large variety of β-lactam antibiotics. In particular, thepresent invention provides engineered PGAs that are capable of removingthe A1/B1/B29 tri-phenyl acetate protecting groups to release freeinsulin.

Generally, naturally occurring PGAs are a heterodimeric enzyme composedof an alpha subunit and a beta-subunit. Wild-type PGA is naturallysynthesized as a pre-pro-PGA polypeptide, containing an N-terminalsignal peptide that mediates translocation to the periplasm and a linkerregion connecting the C-terminus of the alpha subunit to the N-terminusof the beta subunit. Proteolytic processing leads to the matureheterodimeric enzyme. The intermolecular linker region can also functionin promoting proper folding of the enzyme. The PGAs in the presentdisclosure are based on the PGA from Kluyvera citrophila in whichvarious modifications have been introduced to generate improvedenzymatic properties as described in detail below.

For the descriptions provided herein, the use of the singular includesthe plural (and vice versa) unless specifically stated otherwise. Forinstance, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly indicates otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting. It isto be further understood that where descriptions of various embodimentsuse the term “comprising,” those skilled in the art would understandthat in some specific instances, an embodiment can be alternativelydescribed using language “consisting essentially of” or “consisting of.”

Both the foregoing general description, including the drawings, and thefollowing detailed description are exemplary and explanatory only andare not restrictive of this disclosure. Moreover, the section headingsused herein are for organizational purposes only and not to be construedas limiting the subject matter described.

DEFINITIONS

As used herein, the following terms are intended to have the followingmeanings.

In reference to the present disclosure, the technical and scientificterms used in the descriptions herein will have the meanings commonlyunderstood by one of ordinary skill in the art, unless specificallydefined otherwise. Accordingly, the following terms are intended to havethe following meanings. All patents and publications, including allsequences disclosed within such patents and publications, referred toherein are expressly incorporated by reference. Unless otherwiseindicated, the practice of the present invention involves conventionaltechniques commonly used in molecular biology, fermentation,microbiology, and related fields, which are known to those of skill inthe art. Unless defined otherwise herein, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention, thepreferred methods and materials are described. Indeed, it is intendedthat the present invention not be limited to the particular methodology,protocols, and reagents described herein, as these may vary, dependingupon the context in which they are used. The headings provided hereinare not limitations of the various aspects or embodiments of the presentinvention.

Nonetheless, in order to facilitate understanding of the presentinvention, a number of terms are defined below. Numeric ranges areinclusive of the numbers defining the range. Thus, every numerical rangedisclosed herein is intended to encompass every narrower numerical rangethat falls within such broader numerical range, as if such narrowernumerical ranges were all expressly written herein. It is also intendedthat every maximum (or minimum) numerical limitation disclosed hereinincludes every lower (or higher) numerical limitation, as if such lower(or higher) numerical limitations were expressly written herein.

As used herein, the term “comprising” and its cognates are used in theirinclusive sense (i.e., equivalent to the term “including” and itscorresponding cognates).

As used herein and in the appended claims, the singular “a”, “an” and“the” include the plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to a “host cell” includes aplurality of such host cells.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation and amino acid sequences are written left to rightin amino to carboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention that can be had by reference to thespecification as a whole. Accordingly, the terms defined below are morefully defined by reference to the specification as a whole.

As used herein, the terms “protein,” “polypeptide,” and “peptide” areused interchangeably herein to denote a polymer of at least two aminoacids covalently linked by an amide bond, regardless of length orpost-translational modification (e.g., glycosylation, phosphorylation,lipidation, myristilation, ubiquitination, etc.). Included within thisdefinition are D- and L-amino acids, and mixtures of D- and L-aminoacids.

As used herein, “polynucleotide” and “nucleic acid” refer to two or morenucleosides that are covalently linked together. The polynucleotide maybe wholly comprised ribonucleosides (i.e., an RNA), wholly comprised of2′ deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2′deoxyribonucleosides. While the nucleosides will typically be linkedtogether via standard phosphodiester linkages, the polynucleotides mayinclude one or more non-standard linkages. The polynucleotide may besingle-stranded or double-stranded, or may include both single-strandedregions and double-stranded regions. Moreover, while a polynucleotidewill typically be composed of the naturally occurring encodingnucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), itmay include one or more modified and/or synthetic nucleobases (e.g.,inosine, xanthine, hypoxanthine, etc.). Preferably, such modified orsynthetic nucleobases will be encoding nucleobases.

As used herein, “hybridization stringency” relates to hybridizationconditions, such as washing conditions, in the hybridization of nucleicacids. Generally, hybridization reactions are performed under conditionsof lower stringency, followed by washes of varying but higherstringency. The term “moderately stringent hybridization” refers toconditions that permit target-DNA to bind a complementary nucleic acidthat has about 60% identity, preferably about 75% identity, about 85%identity to the target DNA; with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5×SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are known to those of skill in the art.

As used herein, “coding sequence” refers to that portion of a nucleicacid (e.g., a gene) that encodes an amino acid sequence of a protein.

As used herein, “codon optimized” refers to changes in the codons of thepolynucleotide encoding a protein to those preferentially used in aparticular organism such that the encoded protein is efficientlyexpressed in the organism of interest. In some embodiments, thepolynucleotides encoding the PGA enzymes may be codon optimized foroptimal production from the host organism selected for expression.Although the genetic code is degenerate in that most amino acids arerepresented by several codons, called “synonyms” or “synonymous” codons,it is well known that codon usage by particular organisms is nonrandomand biased towards particular codon triplets. This codon usage bias maybe higher in reference to a given gene, genes of common function orancestral origin, highly expressed proteins versus low copy numberproteins, and the aggregate protein coding regions of an organism'sgenome. In some embodiments, the polynucleotides encoding the PGAsenzymes may be codon optimized for optimal production from the hostorganism selected for expression.

As used herein, “preferred, optimal, high codon usage bias codons”refers interchangeably to codons that are used at higher frequency inthe protein coding regions than other codons that code for the sameamino acid. The preferred codons may be determined in relation to codonusage in a single gene, a set of genes of common function or origin,highly expressed genes, the codon frequency in the aggregate proteincoding regions of the whole organism, codon frequency in the aggregateprotein coding regions of related organisms, or combinations thereof.Codons whose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (See e.g., GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney,Bioinform., 14:372-73 [1998]; Stenico et al., Nucleic Acids Res.,222:437-46 [1994]; and Wright, Gene 87:23-29 [1990]). Codon usage tablesare available for a growing list of organisms (See e.g., Wada et al.,Nucleic Acids Res., 20:2111-2118 [1992]; Nakamura et al., Nucl. AcidsRes., 28:292 [2000]; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” Neidhardt, et al. (eds.), ASM Press,Washington D.C., [1996], p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (ESTS), or predictedcoding regions of genomic sequences (See e.g., Uberbacher, Meth.Enzymol., 266:259-281 [1996]; Tiwari et al., Comput. Appl. Biosci.,13:263-270 [1997]).

As used herein, “control sequence” is defined herein to include allcomponents, which are necessary or advantageous for the expression of apolynucleotide and/or polypeptide of the present disclosure. Eachcontrol sequence may be native or foreign to the polynucleotide ofinterest. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator.

As used herein, “operably linked” is defined herein as a configurationin which a control sequence is appropriately placed (i.e., in afunctional relationship) at a position relative to a polynucleotide ofinterest such that the control sequence directs or regulates theexpression of the polynucleotide and/or polypeptide of interest.

As used herein, “promoter sequence” refers to a nucleic acid sequencethat is recognized by a host cell for expression of a polynucleotide ofinterest, such as a coding sequence. The control sequence may comprisean appropriate promoter sequence. The promoter sequence containstranscriptional control sequences, which mediate the expression of apolynucleotide of interest. The promoter may be any nucleic acidsequence which shows transcriptional activity in the host cell of choiceincluding mutant, truncated, and hybrid promoters, and may be obtainedfrom genes encoding extracellular or intracellular polypeptides eitherhomologous or heterologous to the host cell.

As used herein, “naturally occurring” or “wild-type” refers to the formfound in nature. For example, a naturally occurring or wild-typepolypeptide or polynucleotide sequence is a sequence present in anorganism that can be isolated from a source in nature and which has notbeen intentionally modified by human manipulation.

As used herein, “non-naturally occurring,” “engineered,” and“recombinant” when used in the present disclosure with reference to(e.g., a cell, nucleic acid, or polypeptide), refers to a material, or amaterial corresponding to the natural or native form of the material,that has been modified in a manner that would not otherwise exist innature. In some embodiments the material is identical to naturallyoccurring material, but is produced or derived from synthetic materialsand/or by manipulation using recombinant techniques. Non-limitingexamples include, among others, recombinant cells expressing genes thatare not found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise expressed at a different level.

As used herein, “percentage of sequence identity,” “percent identity,”and “percent identical” refer to comparisons between polynucleotidesequences or polypeptide sequences, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whicheither the identical nucleic acid base or amino acid residue occurs inboth sequences or a nucleic acid base or amino acid residue is alignedwith a gap to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. Determination of optimal alignment and percentsequence identity is performed using the BLAST and BLAST 2.0 algorithms(See e.g., Altschul et al., J. Mol. Biol. 215: 403-410 [1990]; andAltschul et al., Nucleic Acids Res. 3389-3402 [1977]). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length Win the querysequence, which either match or satisfy some positive-valued thresholdscore T when aligned with a word of the same length in a databasesequence. T is referred to as, the neighborhood word score threshold(Altschul et al., supra). These initial neighborhood word hits act asseeds for initiating searches to find longer HSPs containing them. Theword hits are then extended in both directions along each sequence foras far as the cumulative alignment score can be increased. Cumulativescores are calculated using, for nucleotide sequences, the parameters M(reward score for a pair of matching residues; always >0) and N (penaltyscore for mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (See e.g., Henikoff and Henikoff, Proc Natl Acad Sci USA 89:10915[1989]).

Numerous other algorithms are available and known in the art thatfunction similarly to BLAST in providing percent identity for twosequences. Optimal alignment of sequences for comparison can beconducted using any suitable method known in the art (e.g., by the localhomology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 [1981];by the homology alignment algorithm of Needleman and Wunsch, J. Mol.Biol. 48:443 [1970]; by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; and/or bycomputerized implementations of these algorithms [GAP, BESTFIT, FASTA,and TFASTA in the GCG Wisconsin Software Package]), or by visualinspection, using methods commonly known in the art. Additionally,determination of sequence alignment and percent sequence identity canemploy the BESTFIT or GAP programs in the GCG Wisconsin Software package(Accelrys, Madison Wis.), using the default parameters provided.

As used herein, “substantial identity” refers to a polynucleotide orpolypeptide sequence that has at least 80 percent sequence identity, atleast 85 percent identity and 89 to 95 percent sequence identity, moreusually at least 99 percent sequence identity as compared to a referencesequence over a comparison window of at least 20 residue positions,frequently over a window of at least 30-50 residues, wherein thepercentage of sequence identity is calculated by comparing the referencesequence to a sequence that includes deletions or additions which total20 percent or less of the reference sequence over the window ofcomparison. In specific embodiments applied to polypeptides, the term“substantial identity” means that two polypeptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 80 percent sequence identity, preferably atleast 89 percent sequence identity, at least 95 percent sequenceidentity or more (e.g., 99 percent sequence identity). In some preferredembodiments, residue positions that are not identical differ byconservative amino acid substitutions.

As used herein, “reference sequence” refers to a defined sequence towhich another sequence is compared. A reference sequence may be a subsetof a larger sequence, for example, a segment of a full-length gene orpolypeptide sequence. Generally, a reference sequence is at least 20nucleotide or amino acid residues in length, at least 25 residues inlength, at least 50 residues in length, or the full length of thenucleic acid or polypeptide. Since two polynucleotides or polypeptidesmay each (1) comprise a sequence (i.e., a portion of the completesequence) that is similar between the two sequences, and (2) may furthercomprise a sequence that is divergent between the two sequences,sequence comparisons between two (or more) polynucleotides orpolypeptide are typically performed by comparing sequences of the twopolynucleotides over a comparison window to identify and compare localregions of sequence similarity. The term “reference sequence” is notintended to be limited to wild-type sequences, and can includeengineered or altered sequences. For example, in some embodiments, a“reference sequence” can be a previously engineered or altered aminoacid sequence.

As used herein, “comparison window” refers to a conceptual segment of atleast about 20 contiguous nucleotide positions or amino acids residueswherein a sequence may be compared to a reference sequence of at least20 contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

As used herein, “corresponding to,” “reference to,” and “relative to”when used in the context of the numbering of a given amino acid orpolynucleotide sequence refers to the numbering of the residues of aspecified reference sequence when the given amino acid or polynucleotidesequence is compared to the reference sequence. In other words, theresidue number or residue position of a given polymer is designated withrespect to the reference sequence rather than by the actual numericalposition of the residue within the given amino acid or polynucleotidesequence. For example, a given amino acid sequence, such as that of anengineered PGA, can be aligned to a reference sequence by introducinggaps to optimize residue matches between the two sequences. In thesecases, although the gaps are present, the numbering of the residue inthe given amino acid or polynucleotide sequence is made with respect tothe reference sequence to which it has been aligned. As used herein, areference to a residue position, such as “Xn” as further describedbelow, is to be construed as referring to “a residue corresponding to”,unless specifically denoted otherwise. Thus, for example, “X94” refersto any amino acid at position 94 in a polypeptide sequence.

As used herein, “improved enzyme property” refers to a PGA that exhibitsan improvement in any enzyme property as compared to a reference PGA.For the engineered PGA polypeptides described herein, the comparison isgenerally made to the wild-type PGA enzyme, although in someembodiments, the reference PGA can be another improved engineered PGA.Enzyme properties for which improvement is desirable include, but arenot limited to, enzymatic activity (which can be expressed in terms ofpercent conversion of the substrate at a specified reaction time using aspecified amount of PGA), thermal stability, solvent stability, pHactivity profile, cofactor requirements, refractoriness to inhibitors(e.g., product inhibition), stereo specificity, and stereo selectivity(including enantioselectivity).

As used herein, “increased enzymatic activity” refers to an improvedproperty of the engineered PGA polypeptides, which can be represented byan increase in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount of PGA) ascompared to the reference PGA enzyme. Exemplary methods to determineenzyme activity are provided in the Examples. Any property relating toenzyme activity may be affected, including the classical enzymeproperties of K_(m), V_(max) or k_(cat), changes of which can lead toincreased enzymatic activity. Improvements in enzyme activity can befrom about 1.5 times the enzymatic activity of the correspondingwild-type PGA enzyme, to as much as 2 times. 5 times, 10 times, 20times, 25 times, 50 times, 75 times, 100 times, or more enzymaticactivity than the naturally occurring PGA or another engineered PGA fromwhich the PGA polypeptides were derived. In specific embodiments, theengineered PGA enzyme exhibits improved enzymatic activity in the rangeof 1.5 to 50 times, 1.5 to 100 times greater than that of the parent PGAenzyme. It is understood by the skilled artisan that the activity of anyenzyme is diffusion limited such that the catalytic turnover rate cannotexceed the diffusion rate of the substrate, including any requiredcofactors. The theoretical maximum of the diffusion limit, ork_(cat)/K_(m), is generally about 10⁸ to 10⁹ (M⁻¹ s⁻¹). Hence, anyimprovements in the enzyme activity of the PGA will have an upper limitrelated to the diffusion rate of the substrates acted on by the PGAenzyme. PGA activity can be measured by any one of standard assays usedfor measuring the release of phenylacetic acid upon cleavage ofpenicillin G, such as by titration (See e.g., Simons and Gibson,Biotechnol. Tech., 13:365-367 [1999]). In some embodiments, the PGAactivity can be measured by using 6-nitrophenylacetamido benzoic acid(NIPAB), which cleavage product 5-amino-2-nitro-benzoic acid isdetectable spectrophotometrically (λmax=405 nm). Comparisons of enzymeactivities are made using a defined preparation of enzyme, a definedassay under a set condition, and one or more defined substrates, asfurther described in detail herein. Generally, when lysates arecompared, the numbers of cells and the amount of protein assayed aredetermined as well as use of identical expression systems and identicalhost cells to minimize variations in amount of enzyme produced by thehost cells and present in the lysates.

As used herein, “increased enzymatic activity” and “increased activity”refer to an improved property of an engineered enzyme, which can berepresented by an increase in specific activity (e.g., productproduced/time/weight protein) or an increase in percent conversion ofthe substrate to the product (e.g., percent conversion of startingamount of substrate to product in a specified time period using aspecified amount of PGA) as compared to a reference enzyme as describedherein. Any property relating to enzyme activity may be affected,including the classical enzyme properties of K_(m), V_(max) or k_(cat),changes of which can lead to increased enzymatic activity. In someembodiments, the PGA enzymes provided herein frees insulin by removingtri-phenyl acetate protecting groups from specific residues of insulin.Comparisons of enzyme activities are made using a defined preparation ofenzyme, a defined assay under a set condition, and one or more definedsubstrates, as further described in detail herein. Generally, whenenzymes in cell lysates are compared, the numbers of cells and theamount of protein assayed are determined as well as use of identicalexpression systems and identical host cells to minimize variations inamount of enzyme produced by the host cells and present in the lysates.

As used herein, “conversion” refers to the enzymatic transformation of asubstrate to the corresponding product.

As used herein “percent conversion” refers to the percent of thesubstrate that is converted to the product within a period of time underspecified conditions. Thus, for example, the “enzymatic activity” or“activity” of a PGA polypeptide can be expressed as “percent conversion”of the substrate to the product.

As used herein, “chemoselectivity” refers to the preferential formationin a chemical or enzymatic reaction of one product over another.

As used herein, “thermostable” and “thermal stable” are usedinterchangeably to refer to a polypeptide that is resistant toinactivation when exposed to a set of temperature conditions (e.g.,40-80° C.) for a period of time (e.g., 0.5-24 hrs) compared to theuntreated enzyme, thus retaining a certain level of residual activity(e.g., more than 60% to 80%) after exposure to elevated temperatures.

As used herein, “solvent stable” refers to the ability of a polypeptideto maintain similar activity (e.g., more than e.g., 60% to 80%) afterexposure to varying concentrations (e.g., 5-99%) of solvent (e.g.,isopropyl alcohol, tetrahydrofuran, 2-methyltetrahydrofuran, acetone,toluene, butylacetate, methyl tert-butylether, etc.) for a period oftime (e.g., 0.5-24 hrs) compared to the untreated enzyme.

As used herein, “pH stable” refers to a PGA polypeptide that maintainssimilar activity (e.g., more than 60% to 80%) after exposure to high orlow pH (e.g., 4.5-6 or 8 to 12) for a period of time (e.g., 0.5-24 hrs)compared to the untreated enzyme.

As used herein, “thermo- and solvent stable” refers to a PGA polypeptidethat is both thermostable and solvent stable.

As used herein, “hydrophilic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity of lessthan zero according to the normalized consensus hydrophobicity scale ofEisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142 [1984]).Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser(S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q), L-Asp (D), L-Lys (K)and L-Arg (R).

As used herein, “acidic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pK value of lessthan about 6 when the amino acid is included in a peptide orpolypeptide. Acidic amino acids typically have negatively charged sidechains at physiological pH due to loss of a hydrogen ion. Geneticallyencoded acidic amino acids include L-Glu (E) and L-Asp (D).

As used herein, “basic amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain exhibiting a pK value ofgreater than about 6 when the amino acid is included in a peptide orpolypeptide. Basic amino acids typically have positively charged sidechains at physiological pH due to association with hydronium ion.Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

As used herein, “polar amino acid or residue” refers to a hydrophilicamino acid or residue having a side chain that is uncharged atphysiological pH, but which has at least one bond in which the pair ofelectrons shared in common by two atoms is held more closely by one ofthe atoms. Genetically encoded polar amino acids include L-Asn (N),L-Gln (Q), L-Ser (S) and L-Thr (T).

As used herein, “hydrophobic amino acid or residue” refers to an aminoacid or residue having a side chain exhibiting a hydrophobicity ofgreater than zero according to the normalized consensus hydrophobicityscale of Eisenberg et al., (Eisenberg et al., J. Mol. Biol., 179:125-142[1984]). Genetically encoded hydrophobic amino acids include L-Pro (P),L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala(A) and L-Tyr (Y).

As used herein, “aromatic amino acid or residue” refers to a hydrophilicor hydrophobic amino acid or residue having a side chain that includesat least one aromatic or heteroaromatic ring. Genetically encodedaromatic amino acids include L-Phe (F), L-Tyr (Y) and L-Trp (W).Although owing to the pKa of its heteroaromatic nitrogen atom L-His (H)it is sometimes classified as a basic residue, or as an aromatic residueas its side chain includes a heteroaromatic ring, herein histidine isclassified as a hydrophilic residue or as a “constrained residue” (seebelow).

As used herein, “constrained amino acid or residue” refers to an aminoacid or residue that has a constrained geometry. Herein, constrainedresidues include L-Pro (P) and L-His (H). Histidine has a constrainedgeometry because it has a relatively small imidazole ring. Proline has aconstrained geometry because it also has a five membered ring.

As used herein, “non-polar amino acid or residue” refers to ahydrophobic amino acid or residue having a side chain that is unchargedat physiological pH and which has bonds in which the pair of electronsshared in common by two atoms is generally held equally by each of thetwo atoms (i.e., the side chain is not polar). Genetically encodednon-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V), L-Ile(I), L-Met (M) and L-Ala (A).

As used herein, “aliphatic amino acid or residue” refers to ahydrophobic amino acid or residue having an aliphatic hydrocarbon sidechain. Genetically encoded aliphatic amino acids include L-Ala (A),L-Val (V), L-Leu (L) and L-Ile (I).

It is noted that cysteine (or “L-Cys” or “[C]”) is unusual in that itcan form disulfide bridges with other L-Cys (C) amino acids or othersulfanyl- or sulfhydryl-containing amino acids. The “cysteine-likeresidues” include cysteine and other amino acids that contain sulfhydrylmoieties that are available for formation of disulfide bridges. Theability of L-Cys (C) (and other amino acids with —SH containing sidechains) to exist in a peptide in either the reduced free —SH or oxidizeddisulfide-bridged form affects whether L-Cys (C) contributes nethydrophobic or hydrophilic character to a peptide. While L-Cys (C)exhibits a hydrophobicity of 0.29 according to the normalized consensusscale of Eisenberg (Eisenberg et al., 1984, supra), it is to beunderstood that for purposes of the present disclosure L-Cys (C) iscategorized into its own unique group.

As used herein, “small amino acid or residue” refers to an amino acid orresidue having a side chain that is composed of a total three or fewercarbon and/or heteroatoms (excluding the α-carbon and hydrogens). Thesmall amino acids or residues may be further categorized as aliphatic,non-polar, polar or acidic small amino acids or residues, in accordancewith the above definitions. Genetically-encoded small amino acidsinclude L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T)and L-Asp (D).

As used herein, “hydroxyl-containing amino acid or residue” refers to anamino acid containing a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

As used herein, “amino acid difference” and “residue difference” referto a difference in the amino acid residue at a position of a polypeptidesequence relative to the amino acid residue at a corresponding positionin a reference sequence. The positions of amino acid differencesgenerally are referred to herein as “Xn,” where n refers to thecorresponding position in the reference sequence upon which the residuedifference is based. For example, a “residue difference at position X40as compared to SEQ ID NO:2” refers to a difference of the amino acidresidue at the polypeptide position corresponding to position 40 of SEQID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has ahistidine at position 40, then a “residue difference at position X40 ascompared to SEQ ID NO:2” refers to an amino acid substitution of anyresidue other than histidine at the position of the polypeptidecorresponding to position 40 of SEQ ID NO:2. In most instances herein,the specific amino acid residue difference at a position is indicated as“XnY” where “Xn” specified the corresponding position as describedabove, and “Y” is the single letter identifier of the amino acid foundin the engineered polypeptide (i.e., the different residue than in thereference polypeptide). In some instances, the present disclosure alsoprovides specific amino acid differences denoted by the conventionalnotation “AnB”, where A is the single letter identifier of the residuein the reference sequence, “n” is the number of the residue position inthe reference sequence, and B is the single letter identifier of theresidue substitution in the sequence of the engineered polypeptide. Insome instances, a polypeptide of the present disclosure can include oneor more amino acid residue differences relative to a reference sequence,which is indicated by a list of the specified positions where residuedifferences are present relative to the reference sequence. In someembodiments, where more than one amino acid can be used in a specificresidue position of a polypeptide, the various amino acid residues thatcan be used are separated by a “/” (e.g., X192A/G). The presentdisclosure includes engineered polypeptide sequences comprising one ormore amino acid differences that include either/or both conservative andnon-conservative amino acid substitutions. The amino acid sequences ofthe specific recombinant carbonic anhydrase polypeptides included in theSequence Listing of the present disclosure include an initiatingmethionine (M) residue (i.e., M represents residue position 1). Theskilled artisan, however, understands that this initiating methionineresidue can be removed by biological processing machinery, such as in ahost cell or in vitro translation system, to generate a mature proteinlacking the initiating methionine residue, but otherwise retaining theenzyme's properties. Consequently, the term “amino acid residuedifference relative to SEQ ID NO:2 at position Xn” as used herein mayrefer to position “Xn” or to the corresponding position (e.g., position(X−1)n) in a reference sequence that has been processed so as to lackthe starting methionine.

As used herein, the phrase “conservative amino acid substitutions”refers to the interchangeability of residues having similar side chains,and thus typically involves substitution of the amino acid in thepolypeptide with amino acids within the same or similar defined class ofamino acids. By way of example and not limitation, in some embodiments,an amino acid with an aliphatic side chain is substituted with anotheraliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine);an amino acid with a hydroxyl side chain is substituted with anotheramino acid with a hydroxyl side chain (e.g., serine and threonine); anamino acids having aromatic side chains is substituted with anotheramino acid having an aromatic side chain (e.g., phenylalanine, tyrosine,tryptophan, and histidine); an amino acid with a basic side chain issubstituted with another amino acid with a basis side chain (e.g.,lysine and arginine); an amino acid with an acidic side chain issubstituted with another amino acid with an acidic side chain (e.g.,aspartic acid or glutamic acid); and/or a hydrophobic or hydrophilicamino acid is replaced with another hydrophobic or hydrophilic aminoacid, respectively. Exemplary conservative substitutions are provided inTable 1.

TABLE 1 Exemplary Conservative Amino Acid Substitutions ResiduePotential Conservative Substitutions A, L, V, I Other aliphatic (A, L,V, I) Other non-polar (A, L, V, I, G, M) G, M Other non-polar (A, L, V,I, G, M) D, E Other acidic (D, E) K, R Other basic (K, R) N, Q, S, TOther polar H, Y, W, F Other aromatic (H, Y, W, F) C, P Non-polar

As used herein, the phrase “non-conservative substitution” refers tosubstitution of an amino acid in the polypeptide with an amino acid withsignificantly differing side chain properties. Non-conservativesubstitutions may use amino acids between, rather than within, thedefined groups and affects (a) the structure of the peptide backbone inthe area of the substitution (e.g., proline for glycine) (b) the chargeor hydrophobicity, or (c) the bulk of the side chain. By way of exampleand not limitation, an exemplary non-conservative substitution can be anacidic amino acid substituted with a basic or aliphatic amino acid; anaromatic amino acid substituted with a small amino acid; and ahydrophilic amino acid substituted with a hydrophobic amino acid.

As used herein, “deletion” refers to modification of the polypeptide byremoval of one or more amino acids from the reference polypeptide.Deletions can comprise removal of 1 or more amino acids, 2 or more aminoacids, 5 or more amino acids, 10 or more amino acids, 15 or more aminoacids, or 20 or more amino acids, up to 10% of the total number of aminoacids, or up to 20% of the total number of amino acids making up thepolypeptide while retaining enzymatic activity and/or retaining theimproved properties of an engineered enzyme. Deletions can be directedto the internal portions and/or terminal portions of the polypeptide. Invarious embodiments, the deletion can comprise a continuous segment orcan be discontinuous.

As used herein, “insertion” refers to modification of the polypeptide byaddition of one or more amino acids to the reference polypeptide. Insome embodiments, the improved engineered PGA enzymes compriseinsertions of one or more amino acids to the naturally occurring PGApolypeptide as well as insertions of one or more amino acids toengineered PGA polypeptides. Insertions can be in the internal portionsof the polypeptide, or to the carboxy or amino terminus Insertions asused herein include fusion proteins as is known in the art. Theinsertion can be a contiguous segment of amino acids or separated by oneor more of the amino acids in the naturally occurring polypeptide.

The term “amino acid substitution set” or “substitution set” refers to agroup of amino acid substitutions in a polypeptide sequence, as comparedto a reference sequence. A substitution set can have 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. Insome embodiments, a substitution set refers to the set of amino acidsubstitutions that is present in any of the variant PGAs listed in theTables provided in the Examples.

As used herein, “fragment” refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can typically have about 80%, about 90%, about 95%,about 98%, or about 99% of the full-length PGA polypeptide, for examplethe polypeptide of SEQ ID NO:2. In some embodiments, the fragment is“biologically active” (i.e., it exhibits the same enzymatic activity asthe full-length sequence).

As used herein, “isolated polypeptide” refers to a polypeptide that issubstantially separated from other contaminants that naturally accompanyit, e.g., protein, lipids, and polynucleotides. The term embracespolypeptides which have been removed or purified from theirnaturally-occurring environment or expression system (e.g., host cell orin vitro synthesis). The improved PGA enzymes may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, theengineered PGA polypeptides of the present disclosure can be an isolatedpolypeptide.

As used herein, “substantially pure polypeptide” refers to a compositionin which the polypeptide species is the predominant species present(i.e., on a molar or weight basis it is more abundant than any otherindividual macromolecular species in the composition), and is generallya substantially purified composition when the object species comprisesat least about 50 percent of the macromolecular species present by moleor % weight. Generally, a substantially pure engineered PGA polypeptidecomposition comprises about 60% or more, about 70% or more, about 80% ormore, about 90% or more, about 91% or more, about 92% or more, about 93%or more, about 94% or more, about 95% or more, about 96% or more, about97% or more, about 98% or more, or about 99% of all macromolecularspecies by mole or % weight present in the composition. Solvent species,small molecules (<500 Daltons), and elemental ion species are notconsidered macromolecular species. In some embodiments, the isolatedimproved PGA polypeptide is a substantially pure polypeptidecomposition.

As used herein, when used in reference to a nucleic acid or polypeptide,the term “heterologous” refers to a sequence that is not normallyexpressed and secreted by an organism (e.g., a wild-type organism). Insome embodiments, the term encompasses a sequence that comprises two ormore subsequences which are not found in the same relationship to eachother as normally found in nature, or is recombinantly engineered sothat its level of expression, or physical relationship to other nucleicacids or other molecules in a cell, or structure, is not normally foundin nature. For instance, a heterologous nucleic acid is typicallyrecombinantly produced, having two or more sequences from unrelatedgenes arranged in a manner not found in nature (e.g., a nucleic acidopen reading frame (ORF) of the invention operatively linked to apromoter sequence inserted into an expression cassette, such as avector). In some embodiments, “heterologous polynucleotide” refers toany polynucleotide that is introduced into a host cell by laboratorytechniques, and includes polynucleotides that are removed from a hostcell, subjected to laboratory manipulation, and then reintroduced into ahost cell.

As used herein, “suitable reaction conditions” refer to those conditionsin the biocatalytic reaction solution (e.g., ranges of enzyme loading,substrate loading, cofactor loading, temperature, pH, buffers,co-solvents, etc.) under which a PGA polypeptide of the presentdisclosure is capable of releasing free insulin by removing tri-phenylacetate protecting groups. Exemplary “suitable reaction conditions” areprovided in the present disclosure and illustrated by the Examples.

As used herein, “loading,” such as in “compound loading,” “enzymeloading,” or “cofactor loading” refers to the concentration or amount ofa component in a reaction mixture at the start of the reaction.

As used herein, “substrate” in the context of a biocatalyst mediatedprocess refers to the compound or molecule acted on by the biocatalyst.

As used herein “product” in the context of a biocatalyst mediatedprocess refers to the compound or molecule resulting from the action ofthe biocatalyst.

As used herein, “equilibration” as used herein refers to the processresulting in a steady state concentration of chemical species in achemical or enzymatic reaction (e.g., interconversion of two species Aand B), including interconversion of stereoisomers, as determined by theforward rate constant and the reverse rate constant of the chemical orenzymatic reaction.

As used herein “acylase” and “acyltransferases” are used interchangeablyto refer to enzymes that are capable of transferring an acyl group froma donor to an acceptor to form esters or amides. The acylase mediatedreverse reaction results in hydrolysis of the ester or amide.

As used herein, “penicillin G” and “benzylpenicillin” refer to theantibiotic also known as(2S,5R,6R)-3,3-dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid (C₁₆H₁₈N₂O₄S). It is primarily effective against Gram-positiveorganisms, although some Gram-negative organisms are also susceptible toit.

As used herein, “penicillin G acylase” and “PGA” are usedinterchangeably to refer to an enzyme having the capability of mediatingcleavage of penicillin G (benzylpenicillin) to phenylacetic acid (PHA)and 6-aminopenicillanic acid (6-APA). In some embodiments, PGA activitycan be based on cleavage of model substrates, for instance the cleavageof 6-nitro-3-(phenylacetamide)benzoic acid to phenylacetic acid and5-amino-2-nitro-benzoic acid. PGAs are also capable of carrying out thereverse reaction of transferring an acyl group of an acyl donor to anacyl acceptor. PGAs as used herein include naturally occurring (wildtype) PGAs as well as non-naturally occurring PGA enzymes comprising oneor more engineered polypeptides generated by human manipulation. Thewild-type PGA gene is a heterodimer consisting of alpha subunit (23.8KDa) and beta subunit (62.2 KDa) linked by a spacer region of 54 aminoacids. Due to the presence of the spacer region, an auto-processing stepis required to form the active protein.

As used herein, “acyl donor” refers to that portion of the acylasesubstrate which donates the acyl group to an acyl acceptor to formesters or amides.

As used herein, “acyl acceptor” refers to that portion of the acylasesubstrate which accepts the acyl group of the acyl donor to form estersor amides.

As used herein, “α-chain sequence” refers to an amino acid sequence thatcorresponds to (e.g., has at least 85% identity to) the residues atpositions 27 to 235 of SEQ ID NO: 2. As used herein, a single chainpolypeptide can comprise an “α-chain sequence” and additionalsequence(s).

As used herein, “β-chain sequence” refers to an amino acid sequence thatcorresponds to (e.g., has at least 85% identity to) residues atpositions 290 to 846 of SEQ ID NO:2. As used herein, a single chainpolypeptide can comprise an “β-chain sequence” and additionalsequence(s).

As used herein, “derived from” when used in the context of engineeredPGA enzymes, identifies the originating PGA enzyme, and/or the geneencoding such PGA enzyme, upon which the engineering was based. Forexample, the engineered PGA enzyme of SEQ ID NO: 60 was obtained byartificially evolving, over multiple generations the gene encoding theK. citrophila PGA alpha-chain and beta-chain sequences of SEQ ID NO:2.Thus, this engineered PGA enzyme is “derived from” the naturallyoccurring or wild-type PGA of SEQ ID NO: 2.

As used herein, “insulin” refers to the polypeptide hormone produced bythe beta-cells of the pancreas in normal individuals. Insulin isnecessary for regulating carbohydrate metabolism, by reducing bloodglucose levels. Systematic deficiency of insulin results in diabetes.Insulin is comprised of 51 amino acids and has a molecular weight ofapproximately 5800 daltons. Insulin is comprised of two peptide chains(designated “A” and “B”), containing one intrasubunit and twointersubunit disulfide bonds. The A chain is composed of 21 amino acidsand the B chain is composed of 30 amino acids. The two chains form ahighly ordered structure, with several alpha-helical regions in both theA and B chains. Isolated chains are inactive. In solution, insulin iseither a monomer, dimer, or hexamer. It is hexameric in the highlyconcentrated preparations used for subcutaneous injection, but becomesmonomeric as it is diluted in body fluids. The definition is intended toencompass proinsulin and any purified isolated polypeptide having partor all of the primary structural conformation and at least one of thebiological properties of naturally-occurring insulin. It is furtherintended to encompass natural and synthetically-derived insulin,including glycoforms, as well as analogs (e.g., polypeptides havingdeletions, insertions, and/or substitutions).

Insulin contains three nucleophilic amines that can potentially reactwith a phenylacetate-donor and be deprotected by PGA. These residuesinclude a Lys on the B-chain at position 29 (B29) and two N-terminalfree amines, Gly on the A-chain at position 1 (A1) and Phe on theB-chain at position 1 (B1). The tri-protected insulin (phenyl acetatechemically attached to A1, B1, B29 residues on human insulin). PGA haspreviously been reported to catalyze hydrolysis ofN-phenylacetate-protected peptides and insulin with exclusiveselectivity for the phenylacetate amide bond, leaving the rest of thepeptide bonds of the protein intact (Brtnik et al., Coll. Czech. Chem.Commun., 46 (8), 1983-1989 [1981]; and Wang et al. Biopolym. 25(Suppl.), S109-S114 [1986]).

Penicillin G Acylases

Penicillin acylase was first described from Penicillium chrysogenumWisc. Q176 by Sakaguchi and Murao (Sakaguchi and Murao, J. Agr. Chem.Soc. Jpn., 23:411 [1950]). Penicillin G acylase is a hydrolytic enzymethat acts on the side chains of penicillin G, cephalosporin G, andrelated antibiotics to produce the β-lactam antibiotic intermediates6-amino penicillanic acid and 7-amino des-acetoxy cephalosporanic acid,with phenyl acetic acid as a common by-product. These antibioticintermediates are among the potential building blocks of semi-syntheticantibiotics, such as ampicillin, amoxicillin, cloxacillin, cephalexin,and cefatoxime.

As indicated above, penicillin G acylases (PGA) are characterized by theability to catalyze the hydrolytic cleavage of penicillin G, with aconjugate base of structural formula (I), to 6-amino penicillanic acid,with a conjugate base of structural formula (II), and phenylacetic acidof structural formula (III), as shown in Scheme 1:

While not being bound by theory, substrate specificity appearsassociated with recognition of the hydrophobic phenyl group while anucleophile, which in some PGAs is a serine residue at the N-terminus ofthe beta-chain acts as the acceptor of beta-lactam and a variety ofother groups, such as beta-amino acids. PGAs can also be characterizedby the ability to cleave a model substrates analogous to penicillin G,for instance cleavage of 6-nitro-3-(phenylacetamido)benzoic acid (NIPAB)of structural formula (IV), as shown in Scheme 2:

to phenylacetic acid of structural formula (III) and5-amino-2-nitro-benzoic acid of structural formula (V) (See e.g., Alkemaet al., Anal. Biochem., 275:47-53 [1999]). Because the5-amino-2-nitro-benzoic acid is chromogenic, the substrate of formula(IV) provides a convenient way of measuring PGA activity. In addition tothe foregoing reactions, PGAs can also be used in the kinetic resolutionof DL-tert leucine for the preparation of optically pure tert leucine(See e.g., Liu et al., Prep. Biochem. Biotechnol., 36:235-41 [2006]).

The PGAs of the present disclosure are based on the enzyme obtained fromthe organism Kluyvera citrophila (K. citrophila). As with PGAs fromother organisms, the PGA of K. citrophila is a heterodimeric enzymecomprised of an alpha-subunit and a beta-subunit that is generated byproteolytic processing of a pre-pro-PGA polypeptide. Removal of a signalpeptide and a spacer peptide produces the mature heterodimer (See e.g.,Barbero et al., Gene 49:69-80 [1986]). The amino acid sequence of thenaturally occurring pre-pro-PGA polypeptide of K. citrophila is publiclyavailable (See e.g., Genbank accession No. P07941, [gi:129551]) and isprovided herein as SEQ ID NO:2. The alpha-chain sequence of thenaturally occurring K. citrophila PGA corresponds to residues 27 to 235of SEQ ID NO:2. The beta-chain sequence of the naturally occurring K.citrophila PGA corresponds to residues 290 to 846 of SEQ ID NO:2.Residues 1 to 26 of SEQ ID NO:2 correspond to the signal peptide andresidues 236-289 of SEQ ID NO:2 correspond to the linking propeptide,both of which are removed to generate the naturally occurring mature PGAenzyme which is a heterodimer comprising an α-chain subunit and aβ-chain subunit.

In some embodiments, the present invention provides engineered PGApolypeptides with amino acid sequences that have at least about 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%or more sequence identity to SEQ ID NOS:2, 4, 6, 8, 10 and/or 12.

The present invention provides insulin-specific deacylation biocatalystssuitable for laboratory scale preparative use. Directed evolution wasused to develop efficient acylase variants capable of completelyremoving the A1/B1/B29-tri-phenyl acetate protecting groups on insulinand generate >99% free insulin. Following only two rounds of evolution,variants that generate more than 99% free insulin in less than 6 hrs. at0.8 g/L enzyme loading were produced. The final best variant PGA_005 was˜8-fold improved over the initial backbone. The PGA variants providedherein are capable of accepting a wide range of acyl groups, exhibitincreased solvent stability, and improved thermostability, as comparedto the wild-type PGA. The variant PGAs provided herein lack the spacerregion. Thus, the auto-processing step is not required in order toproduce active enzymes.

The present invention also provides polynucleotides encoding theengineered PGA polypeptides. In some embodiments, the polynucleotidesare operatively linked to one or more heterologous regulatory sequencesthat control gene expression, to create a recombinant polynucleotidecapable of expressing the polypeptide. Expression constructs containinga heterologous polynucleotide encoding the engineered PGA polypeptidescan be introduced into appropriate host cells to express thecorresponding PGA polypeptide.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode the improved PGA enzymesdisclosed herein. Thus, having identified a particular amino acidsequence, those skilled in the art could make any number of differentnucleic acids by simply modifying the sequence of one or more codons ina way which does not change the amino acid sequence of the protein. Inthis regard, the present disclosure specifically contemplates each andevery possible variation of polynucleotides that could be made byselecting combinations based on the possible codon choices, and all suchvariations are to be considered specifically disclosed for anypolypeptide disclosed herein, including the amino acid sequencespresented in the Tables in Examples 5 and 6.

In various embodiments, the codons are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells.

In certain embodiments, all codons need not be replaced to optimize thecodon usage of the PGA polypeptides since the natural sequence willcomprise preferred codons and because use of preferred codons may not berequired for all amino acid residues. Consequently, codon optimizedpolynucleotides encoding the PGA enzymes may contain preferred codons atabout 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions ofthe full length coding region.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding a PGA polypeptide with an amino acid sequence that has at leastabout 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity to the alpha-chain and/orbeta-chain any of the reference engineered PGA polypeptides describedherein. Accordingly, in some embodiments, the polynucleotide encodes anamino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to areference alpha- and beta-chain sequences based on SEQ ID NO:2. In someembodiments, the polynucleotide encodes an alpha- and/or beta-chainamino acid sequence of SEQ ID NO:2.

In some embodiments, the polynucleotide comprises a nucleotide sequenceencoding a PGA polypeptide with an amino acid sequence that has at leastabout 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity to SEQ ID NO:4, 6, 8, 10, and/or12. Accordingly, in some embodiments, the polynucleotide encodes anamino acid sequence that is at least about 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ IDNO:1, 3, 5, 7, 9, and/or 11.

In some embodiments, an isolated polynucleotide encoding an improved PGApolypeptide was manipulated in a variety of ways to provide for improvedactivity and/or expression of the polypeptide. Manipulation of theisolated polynucleotide prior to its insertion into a vector may bedesirable or necessary depending on the expression vector. Thetechniques for modifying polynucleotides and nucleic acid sequencesutilizing recombinant DNA methods are well known in the art.

For example, mutagenesis and directed evolution methods can be readilyapplied to polynucleotides to generate variant libraries that can beexpressed, screened, and assayed. Mutagenesis and directed evolutionmethods are well known in the art (See e.g., U.S. Pat. Nos. 5,605,793,5,830,721, 6,132,970, 6,420,175, 6,277,638, 6,365,408, 6,602,986,7,288,375, 6,287,861, 6,297,053, 6,576,467, 6,444,468, 5,811238,6,117,679, 6,165,793, 6,180,406, 6,291,242, 6,995,017, 6,395,547,6,506,602, 6,519,065, 6,506,603, 6,413,774, 6,573,098, 6,323,030,6,344,356, 6,372,497, 7,868,138, 5,834,252, 5,928,905, 6,489,146,6,096,548, 6,387,702, 6,391,552, 6,358,742, 6,482,647, 6,335,160,6,653,072, 6,355,484, 6,03,344, 6,319,713, 6,613,514, 6,455,253,6,579,678, 6,586,182, 6,406,855, 6,946,296, 7,534,564, 7,776,598,5,837,458, 6,391,640, 6,309,883, 7,105,297, 7,795,030, 6,326,204,6,251,674, 6,716,631, 6,528,311, 6,287,862, 6,335,198, 6,352,859,6,379,964, 7,148,054, 7,629,170, 7,620,500, 6,365,377, 6,358,740,6,406,910, 6,413,745, 6,436,675, 6,961,664, 7,430,477, 7,873,499,7,702,464, 7,783,428, 7,747,391, 7,747,393, 7,751,986, 6,376,246,6,426,224, 6,423,542, 6,479,652, 6,319,714, 6,521,453, 6,368,861,7,421,347, 7,058,515, 7,024,312, 7,620,502, 7,853,410, 7,957,912,7,904,249, and all related non-US counterparts; Ling et al., Anal.Biochem., 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol., 57:369-74[1996]; Smith, Ann. Rev. Genet., 19:423-462 [1985]; Botstein et al.,Science, 229:1193-1201 [1985]; Carter, Biochem. J., 237:1-7 [1986];Kramer et al., Cell, 38:879-887 [1984]; Wells et al., Gene, 34:315-323[1985]; Minshull et al., Curr. Op. Chem. Biol., 3:284-290 [1999];Christians et al., Nat. Biotechnol., 17:259-264 [1999]; Crameri et al.,Nature, 391:288-291 [1998]; Crameri, et al., Nat. Biotechnol.,15:436-438 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A.,94:4504-4509 [1997]; Crameri et al., Nat. Biotechnol., 14:315-319[1996]; Stemmer, Nature, 370:389-391 [1994]; Stemmer, Proc. Nat. Acad.Sci. USA, 91:10747-10751 [1994]; WO 95/22625; WO 97/0078; WO 97/35966;WO 98/27230; WO 00/42651; WO 01/75767; and WO 2009/152336, all of whichare incorporated herein by reference).

In some embodiments, the variant PGA acylases of the present inventionfurther comprise additional sequences that do not alter the encodedactivity of the enzyme. For example, in some embodiments, the variantPGA acylases are linked to an epitope tag or to another sequence usefulin purification.

In some embodiments, the variant PGA acylase polypeptides of the presentinvention are secreted from the host cell in which they are expressed(e.g., a yeast or filamentous fungal host cell) and are expressed as apre-protein including a signal peptide (i.e., an amino acid sequencelinked to the amino terminus of a polypeptide and which directs theencoded polypeptide into the cell secretory pathway).

In some embodiments, the signal peptide is an endogenous K. citrophilaPGA acylase signal peptide. In some other embodiments, signal peptidesfrom other K. citrophila secreted proteins are used. In someembodiments, other signal peptides find use, depending on the host celland other factors. Effective signal peptide coding regions forfilamentous fungal host cells include, but are not limited to, thesignal peptide coding regions obtained from Aspergillus oryzae TAKAamylase, Aspergillus niger neutral amylase, Aspergillus nigerglucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolenscellulase, Humicola lanuginosa lipase, and T. reesei cellobiohydrolaseII. Signal peptide coding regions for bacterial host cells include, butare not limited to the signal peptide coding regions obtained from thegenes for Bacillus NCIB 11837 maltogenic amylase, Bacillusstearothermophilus alpha-amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutralproteases (nprT, nprS, nprM), and Bacillus subtilis prsA. In someadditional embodiments, other signal peptides find use in the presentinvention (See e.g., Simonen and Palva, Microbiol. Rev., 57: 109-137[1993], incorporated herein by reference). Additional useful signalpeptides for yeast host cells include those from the genes forSaccharomyces cerevisiae alpha-factor, Saccharomyces cerevisiae SUC2invertase (See e.g., Taussig and Carlson, Nucl. Acids Res., 11:1943-54[1983]; SwissProt Accession No. P00724; and Romanos et al., Yeast8:423-488 [1992]). In some embodiments, variants of these signalpeptides and other signal peptides find use. Indeed, it is not intendedthat the present invention be limited to any specific signal peptide, asany suitable signal peptide known in the art finds use in the presentinvention.

In some embodiments, the present invention provides polynucleotidesencoding variant PGA acylase polypeptides, and/or biologically activefragments thereof, as described herein. In some embodiments, thepolynucleotide is operably linked to one or more heterologous regulatoryor control sequences that control gene expression to create arecombinant polynucleotide capable of expressing the polypeptide. Insome embodiments, expression constructs containing a heterologouspolynucleotide encoding a variant PGA acylase is introduced intoappropriate host cells to express the variant PGA acylase.

Those of ordinary skill in the art understand that due to the degeneracyof the genetic code, a multitude of nucleotide sequences encodingvariant PGA acylase polypeptides of the present invention exist. Forexample, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode theamino acid arginine. Thus, at every position in the nucleic acids of theinvention where an arginine is specified by a codon, the codon can bealtered to any of the corresponding codons described above withoutaltering the encoded polypeptide. It is understood that “U” in an RNAsequence corresponds to “T” in a DNA sequence. The inventioncontemplates and provides each and every possible variation of nucleicacid sequence encoding a polypeptide of the invention that could be madeby selecting combinations based on possible codon choices.

As indicated above, DNA sequence encoding a PGA may also be designed forhigh codon usage bias codons (codons that are used at higher frequencyin the protein coding regions than other codons that code for the sameamino acid). The preferred codons may be determined in relation to codonusage in a single gene, a set of genes of common function or origin,highly expressed genes, the codon frequency in the aggregate proteincoding regions of the whole organism, codon frequency in the aggregateprotein coding regions of related organisms, or combinations thereof. Acodon whose frequency increases with the level of gene expression istypically an optimal codon for expression. In particular, a DNA sequencecan be optimized for expression in a particular host organism. A varietyof methods are well-known in the art for determining the codon frequency(e.g., codon usage, relative synonymous codon usage) and codonpreference in specific organisms, including multivariate analysis (e.g.,using cluster analysis or correspondence analysis,) and the effectivenumber of codons used in a gene. The data source for obtaining codonusage may rely on any available nucleotide sequence capable of codingfor a protein. These data sets include nucleic acid sequences actuallyknown to encode expressed proteins (e.g., complete protein codingsequences-CDS), expressed sequence tags (ESTs), or predicted codingregions of genomic sequences, as is well-known in the art.Polynucleotides encoding variant PGAs can be prepared using any suitablemethods known in the art. Typically, oligonucleotides are individuallysynthesized, then joined (e.g., by enzymatic or chemical ligationmethods, or polymerase-mediated methods) to form essentially any desiredcontinuous sequence. In some embodiments, polynucleotides of the presentinvention are prepared by chemical synthesis using, any suitable methodsknown in the art, including but not limited to automated syntheticmethods. For example, in the phosphoramidite method, oligonucleotidesare synthesized (e.g., in an automatic DNA synthesizer), purified,annealed, ligated and cloned in appropriate vectors. In someembodiments, double stranded DNA fragments are then obtained either bysynthesizing the complementary strand and annealing the strands togetherunder appropriate conditions, or by adding the complementary strandusing DNA polymerase with an appropriate primer sequence. There arenumerous general and standard texts that provide methods useful in thepresent invention are well known to those skilled in the art.

The engineered PGAs can be obtained by subjecting the polynucleotideencoding the naturally occurring PGA to mutagenesis and/or directedevolution methods, as discussed above. Mutagenesis may be performed inaccordance with any of the techniques known in the art, including randomand site-specific mutagenesis. Directed evolution can be performed withany of the techniques known in the art to screen for improved variantsincluding shuffling. Other directed evolution procedures that find useinclude, but are not limited to staggered extension process (StEP), invitro recombination, mutagenic PCR, cassette mutagenesis, splicing byoverlap extension (SOEing), ProSAR™ directed evolution methods, etc., aswell as any other suitable methods.

The clones obtained following mutagenesis treatment are screened forengineered PGAs having a desired improved enzyme property. Measuringenzyme activity from the expression libraries can be performed using thestandard biochemistry technique of monitoring the rate of productformation. Where an improved enzyme property desired is thermalstability, enzyme activity may be measured after subjecting the enzymepreparations to a defined temperature and measuring the amount of enzymeactivity remaining after heat treatments. Clones containing apolynucleotide encoding a PGA are then isolated, sequenced to identifythe nucleotide sequence changes (if any), and used to express the enzymein a host cell.

When the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis (e.g., using theclassical phosphoramidite method described by Beaucage et al., Tet.Lett., 22:1859-69 [1981], or the method described by Matthes et al.,EMBO J., 3:801-05 [1984], as it is typically practiced in automatedsynthetic methods). According to the phosphoramidite method,oligonucleotides are synthesized (e.g., in an automatic DNAsynthesizer), purified, annealed, ligated and cloned in appropriatevectors. In addition, essentially any nucleic acid can be obtained fromany of a variety of commercial sources (e.g., The Midland CertifiedReagent Company, Midland, Tex., The Great American Gene Company, Ramona,Calif., ExpressGen Inc. Chicago, Ill., Operon Technologies Inc.,Alameda, Calif., and many others).

The present invention also provides recombinant constructs comprising asequence encoding at least one variant PGA, as provided herein. In someembodiments, the present invention provides an expression vectorcomprising a variant PGA polynucleotide operably linked to aheterologous promoter. In some embodiments, expression vectors of thepresent invention are used to transform appropriate host cells to permitthe host cells to express the variant PGA protein. Methods forrecombinant expression of proteins in fungi and other organisms are wellknown in the art, and a number expression vectors are available or canbe constructed using routine methods. In some embodiments, nucleic acidconstructs of the present invention comprise a vector, such as, aplasmid, a cosmid, a phage, a virus, a bacterial artificial chromosome(BAC), a yeast artificial chromosome (YAC), and the like, into which anucleic acid sequence of the invention has been inserted. In someembodiments, polynucleotides of the present invention are incorporatedinto any one of a variety of expression vectors suitable for expressingvariant PGA polypeptide(s). Suitable vectors include, but are notlimited to chromosomal, nonchromosomal and synthetic DNA sequences(e.g., derivatives of SV40), as well as bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, pseudorabies, adenovirus, adeno-associated virus, retroviruses,and many others. Any suitable vector that transduces genetic materialinto a cell, and, if replication is desired, which is replicable andviable in the relevant host finds use in the present invention. In someembodiments, the construct further comprises regulatory sequences,including but not limited to a promoter, operably linked to the proteinencoding sequence. Large numbers of suitable vectors and promoters areknown to those of skill in the art. Indeed, in some embodiments, inorder to obtain high levels of expression in a particular host it isoften useful to express the variant PGAs of the present invention underthe control of a heterologous promoter. In some embodiments, a promotersequence is operably linked to the 5′ region of the variant PGA codingsequence using any suitable method known in the art. Examples of usefulpromoters for expression of variant PGAs include, but are not limited topromoters from fungi. In some embodiments, a promoter sequence thatdrives expression of a gene other than a PGA gene in a fungal strainfinds use. As a non-limiting example, a fungal promoter from a geneencoding an endoglucanase may be used. In some embodiments, a promotersequence that drives the expression of a PGA gene in a fungal strainother than the fungal strain from which the PGAs were derived finds use.Examples of other suitable promoters useful for directing thetranscription of the nucleotide constructs of the present invention in afilamentous fungal host cell include, but are not limited to promotersobtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucormiehei aspartic proteinase, Aspergillus niger neutral alpha-amylase,Aspergillus niger acid stable alpha-amylase, Aspergillus niger orAspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase,Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusariumoxysporum trypsin-like protease (See e.g., WO 96/00787, incorporatedherein by reference), as well as the NA2-tpi promoter (a hybrid of thepromoters from the genes for Aspergillus niger neutral alpha-amylase andAspergillus oryzae triose phosphate isomerase), promoters such as cbh1,cbh2, egl1, egl2, pepA, hfb1, hfb2, xyn1, amy, and glaA (See e.g.,Nunberg et al., Mol. Cell Biol., 4:2306-2315 [1984]; Boel et al., EMBOJ., 3:1581-85 [1984]; and European Patent Appln. 137280, all of whichare incorporated herein by reference), and mutant, truncated, and hybridpromoters thereof.

In yeast host cells, useful promoters include, but are not limited tothose from the genes for Saccharomyces cerevisiae enolase (eno-1),Saccharomyces cerevisiae galactokinase (gall), Saccharomyces cerevisiaealcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase(ADH2/GAP), and S. cerevisiae 3-phosphoglycerate kinase. Additionaluseful promoters useful for yeast host cells are known in the art (Seee.g., Romanos et al., Yeast 8:423-488 [1992], incorporated herein byreference). In addition, promoters associated with chitinase productionin fungi find use in the present invention (See e.g., Blaiseau andLafay, Gene 120243-248 [1992]; and Limon et al., Curr. Genet., 28:478-83[1995], both of which are incorporated herein by reference).

For bacterial host cells, suitable promoters for directing transcriptionof the nucleic acid constructs of the present disclosure, include butare not limited to the promoters obtained from the E. coli lac operon,E. coli trp operon, bacteriophage λ, Streptomyces coelicolor agarasegene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacilluslicheniformis alpha-amylase gene (amyL), Bacillus stearothermophilusmaltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylasegene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillussubtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Seee.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731[1978]), as well as the tac promoter (See e.g., DeBoer et al., Proc.Natl. Acad. Sci. USA 80: 21-25 [1983]).

In some embodiments, cloned variant PGAs of the present invention alsohave a suitable transcription terminator sequence, a sequence recognizedby a host cell to terminate transcription. The terminator sequence isoperably linked to the 3′ terminus of the nucleic acid sequence encodingthe polypeptide. Any terminator that is functional in the host cell ofchoice finds use in the present invention. Exemplary transcriptionterminators for filamentous fungal host cells include, but are notlimited to those obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease (See also, U.S. Pat. No. 7,399,627,incorporated herein by reference). In some embodiments, exemplaryterminators for yeast host cells include those obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are well-known to those skilled in the art (See e.g.,Romanos et al., Yeast 8:423-88 [1992]).

In some embodiments, a suitable leader sequence is part of a clonedvariant PGA sequence, which is a nontranslated region of an mRNA that isimportant for translation by the host cell. The leader sequence isoperably linked to the 5′ terminus of the nucleic acid sequence encodingthe polypeptide. Any leader sequence that is functional in the host cellof choice finds use in the present invention. Exemplary leaders forfilamentous fungal host cells include, but are not limited to thoseobtained from the genes for Aspergillus oryzae TAKA amylase andAspergillus nidulans triose phosphate isomerase. Suitable leaders foryeast host cells include, but are not limited to those obtained from thegenes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomycescerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiaealpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

In some embodiments, the sequences of the present invention alsocomprise a polyadenylation sequence, which is a sequence operably linkedto the 3′ terminus of the nucleic acid sequence and which, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice finds use in the presentinvention. Exemplary polyadenylation sequences for filamentous fungalhost cells include, but are not limited to those obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Fusarium oxysporumtrypsin-like protease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are known in the art (Seee.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990 [1995]).

In some embodiments, the control sequence comprises a signal peptidecoding region encoding an amino acid sequence linked to the aminoterminus of a polypeptide and directs the encoded polypeptide into thecell's secretory pathway. The 5′ end of the coding sequence of thenucleic acid sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region that encodes the secreted polypeptide. Alternatively,the 5′ end of the coding sequence may contain a signal peptide codingregion that is foreign to the coding sequence. The foreign signalpeptide coding region may be required where the coding sequence does notnaturally contain a signal peptide coding region.

Alternatively, the foreign signal peptide coding region may simplyreplace the natural signal peptide coding region in order to enhancesecretion of the polypeptide. However, any signal peptide coding regionwhich directs the expressed polypeptide into the secretory pathway of ahost cell of choice may be used in the present invention.

Effective signal peptide coding regions for bacterial host cellsinclude, but are not limited to the signal peptide coding regionsobtained from the genes for Bacillus NCIB 11837 maltogenic amylase,Bacillus stearothermophilus alpha-amylase, Bacillus licheniformissubtilisin, Bacillus licheniformis beta-lactamase, Bacillusstearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillussubtilis prsA. Further signal peptides are known in the art (See e.g.,Simonen and Palva, Microbiol. Rev., 57: 109-137 [1993]).

Effective signal peptide coding regions for filamentous fungal hostcells include, but are not limited to the signal peptide coding regionsobtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillusniger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor mieheiaspartic proteinase, Humicola insolens cellulase, and Humicolalanuginosa lipase.

Useful signal peptides for yeast host cells include, but are not limitedto genes for Saccharomyces cerevisiae alpha-factor and Saccharomycescerevisiae invertase. Other useful signal peptide coding regions areknown in the art (See e.g., Romanos et al., [1992], supra).

In some embodiments, the control sequence comprises a propeptide codingregion that codes for an amino acid sequence positioned at the aminoterminus of a polypeptide. The resultant polypeptide is known as aproenzyme or propolypeptide (or a zymogen in some cases). Apropolypeptide is generally inactive and can be converted to a matureactive PGA polypeptide by catalytic or autocatalytic cleavage of thepropeptide from the propolypeptide. The propeptide coding region may beobtained from the genes for Bacillus subtilis alkaline protease (aprE),Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiaealpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthorathermophila lactase (See e.g., WO 95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

In some embodiments, regulatory sequences are also used to allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude, but are not limited to the lac, tac, and trp operator systems.In yeast host cells, suitable regulatory systems include, as examples,the ADH2 system or GAL1 system. In filamentous fungi, suitableregulatory sequences include the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the PGA polypeptide ofthe present invention would be operably linked with the regulatorysequence.

Thus, in additional embodiments, the present invention providesrecombinant expression vectors comprising a polynucleotide encoding anengineered PGA polypeptide or a variant thereof, and one or moreexpression regulating regions such as a promoter and a terminator, areplication origin, etc., depending on the type of hosts into which theyare to be introduced. In some embodiments, the various nucleic acid andcontrol sequences described above are joined together to produce arecombinant expression vector that may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, insome embodiments, the nucleic acid sequence are expressed by insertingthe nucleic acid sequence or a nucleic acid construct comprising thesequence into an appropriate vector for expression. In creating theexpression vector, the coding sequence is located in the vector so thatthe coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector comprises any suitable vector (e.g., aplasmid or virus), that can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector typically depends on thecompatibility of the vector with the host cell into which the vector isto be introduced. In some embodiments, the vectors are linear or closedcircular plasmids.

In some embodiments, the expression vector is an autonomouslyreplicating vector (i.e., a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, such as a plasmid, an extrachromosomal element, aminichromosome, or an artificial chromosome). In some embodiments, thevector contains any means for assuring self-replication. Alternatively,in some other embodiments, upon being introduced into the host cell, thevector is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, inadditional embodiments, a single vector or plasmid or two or morevectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon find use.

In some embodiments, the expression vector of the present inventioncontains one or more selectable markers, which permit easy selection oftransformed cells. A “selectable marker” is a gene, the product of whichprovides for biocide or viral resistance, resistance to antimicrobialsor heavy metals, prototrophy to auxotrophs, and the like. Any suitableselectable markers for use in a filamentous fungal host cell find use inthe present invention, including, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Additional markers useful in host cells such as Aspergillus, include butare not limited to the amdS and pyrG genes of Aspergillus nidulans orAspergillus oryzae, and the bar gene of Streptomyces hygroscopicus.Suitable markers for yeast host cells include, but are not limited toADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Examples of bacterialselectable markers include, but are not limited to the dal genes fromBacillus subtilis or Bacillus licheniformis, or markers, which conferantibiotic resistance such as ampicillin, kanamycin, chloramphenicol,and or tetracycline resistance.

In some embodiments, the expression vectors of the present inventioncontain an element(s) that permits integration of the vector into thehost cell's genome or autonomous replication of the vector in the cellindependent of the genome. In some embodiments involving integrationinto the host cell genome, the vectors rely on the nucleic acid sequenceencoding the polypeptide or any other element of the vector forintegration of the vector into the genome by homologous or nonhomologousrecombination.

In some alternative embodiments, the expression vectors containadditional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements preferably contain a sufficient number of nucleicacids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 basepairs, and most preferably 800 to 10,000 base pairs, which are highlyhomologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, pTA1060, or pAMβ1 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes it's functioningtemperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl.Acad. Sci. USA 75:1433 [1978]).

In some embodiments, more than one copy of a nucleic acid sequence ofthe present invention is inserted into the host cell to increaseproduction of the gene product. An increase in the copy number of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome or byincluding an amplifiable selectable marker gene with the nucleic acidsequence where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the nucleic acid sequence,can be selected for by cultivating the cells in the presence of theappropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors include,but are not limited to the p3xFLAG™™ expression vectors (Sigma-AldrichChemicals), which include a CMV promoter and hGH polyadenylation sitefor expression in mammalian host cells and a pBR322 origin ofreplication and ampicillin resistance markers for amplification in E.coli. Other suitable expression vectors include, but are not limited topBluescriptII SK(-) and pBK-CMV (Stratagene), and plasmids derived frompBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4 (Invitrogen) or pPoly(See e.g., Lathe et al., Gene 57:193-201 [1987]).

Thus, in some embodiments, a vector comprising a sequence encoding atleast one variant PGA is transformed into a host cell in order to allowpropagation of the vector and expression of the variant PGA(s). In someembodiments, the variant PGAs are post-translationally modified toremove the signal peptide and in some cases may be cleaved aftersecretion. In some embodiments, the transformed host cell describedabove is cultured in a suitable nutrient medium under conditionspermitting the expression of the variant PGA(s). Any suitable mediumuseful for culturing the host cells finds use in the present invention,including, but not limited to minimal or complex media containingappropriate supplements. In some embodiments, host cells are grown inHTP media. Suitable media are available from various commercialsuppliers or may be prepared according to published recipes (e.g., incatalogues of the American Type Culture Collection).

In another aspect, the present invention provides host cells comprisinga polynucleotide encoding an improved PGA polypeptide provided herein,the polynucleotide being operatively linked to one or more controlsequences for expression of the PGA enzyme in the host cell. Host cellsfor use in expressing the PGA polypeptides encoded by the expressionvectors of the present invention are well known in the art and includebut are not limited to, bacterial cells, such as E. coli, Bacillusmegatarium, Lactobacillus kefir, Streptomyces and Salmonella typhimuriumcells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiaeor Pichia pastoris (ATCC Accession No. 201178)); insect cells such asDrosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS,BHK, 293, and Bowes melanoma cells; and plant cells. Appropriate culturemedia and growth conditions for the above-described host cells are wellknown in the art.

Polynucleotides for expression of the PGA may be introduced into cellsby various methods known in the art. Techniques include among others,electroporation, biolistic particle bombardment, liposome mediatedtransfection, calcium chloride transfection, and protoplast fusion.Various methods for introducing polynucleotides into cells are known tothose skilled in the art.

In some embodiments, the host cell is a eukaryotic cell. Suitableeukaryotic host cells include, but are not limited to, fungal cells,algal cells, insect cells, and plant cells. Suitable fungal host cellsinclude, but are not limited to, Ascomycota, Basidiomycota,Deuteromycota, Zygomycota, Fungi imperfecti. In some embodiments, thefungal host cells are yeast cells and filamentous fungal cells. Thefilamentous fungal host cells of the present invention include allfilamentous forms of the subdivision Eumycotina and Oomycota.Filamentous fungi are characterized by a vegetative mycelium with a cellwall composed of chitin, cellulose and other complex polysaccharides.The filamentous fungal host cells of the present invention aremorphologically distinct from yeast.

In some embodiments of the present invention, the filamentous fungalhost cells are of any suitable genus and species, including, but notlimited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera,Ceriporiopsis, Cephalosporium, Chrysosporium, Cochliobolus, Corynascus,Cryphonectria, Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis,Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora,Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces,Pyricularia, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium,Sporotrichum, Talaromyces, Thermoascus, Thielavia, Trametes,Tolypocladium, Trichoderma, Verticillium, and/or Volvariella, and/orteleomorphs, or anamorphs, and synonyms, basionyms, or taxonomicequivalents thereof.

In some embodiments of the present invention, the host cell is a yeastcell, including but not limited to cells of Candida, Hansenula,Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, or Yarrowiaspecies. In some embodiments of the present invention, the yeast cell isHansenula polymorpha, Saccharomyces cerevisiae, Saccharomycescarlsbergensis, Saccharomyces diastaticus, Saccharomyces norbensis,Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris,Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichiamembranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichiasalictaria, Pichia quercuum, Pichia pijperi, Pichia stipitis, Pichiamethanolica, Pichia angusta, Kluyveromyces lactis, Candida albicans, orYarrowia lipolytica.

In some embodiments of the invention, the host cell is an algal cellsuch as Chlamydomonas (e.g., C. reinhardtii) and Phormidium (P. sp.ATCC29409).

In some other embodiments, the host cell is a prokaryotic cell. Suitableprokaryotic cells include, but are not limited to Gram-positive,Gram-negative and Gram-variable bacterial cells. Any suitable bacterialorganism finds use in the present invention, including but not limitedto Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Acinetobacter,Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium,Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter,Clostridium, Corynebacterium, Chromatium, Coprococcus, Escherichia,Enterococcus, Enterobacter, Erwinia, Fusobacterium, Faecalibacterium,Francisella, Flavobacterium, Geobacillus, Haemophilus, Helicobacter,Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus,Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium,Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus,Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia,Rhodospirillum, Rhodococcus, Scenedesmus, Streptomyces, Streptococcus,Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella,Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula,Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella,Yersinia and Zymomonas. In some embodiments, the host cell is a speciesof Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium,Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium,Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus,Lactococcus, Pantoea, Pseudomonas, Staphylococcus, Salmonella,Streptococcus, Streptomyces, or Zymomonas. In some embodiments, thebacterial host strain is non-pathogenic to humans. In some embodimentsthe bacterial host strain is an industrial strain. Numerous bacterialindustrial strains are known and suitable in the present invention. Insome embodiments of the present invention, the bacterial host cell is anAgrobacterium species (e.g., A. radiobacter, A. rhizogenes, and A.rubi). In some embodiments of the present invention, the bacterial hostcell is an Arthrobacter species (e.g., A. aurescens, A. citreus, A.globformis, A. hydrocarboglutamicus, A. mysorens, A. nicotianae, A.paraffineus, A. protophonniae, A. roseoparqffinus, A. sulfureus, and A.ureafaciens). In some embodiments of the present invention, thebacterial host cell is a Bacillus species (e.g., B. thuringensis, B.anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B.pumilus, B. lautus, B. coagulans, B. brevis, B. firmus, B. alkaophius,B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, andB. amyloliquefaciens). In some embodiments, the host cell is anindustrial Bacillus strain including but not limited to B. subtilis, B.pumilus, B. licheniformis, B. megaterium, B. clausii, B.stearothermophilus, or B. amyloliquefaciens. In some embodiments, theBacillus host cells are B. subtilis, B. licheniformis, B. megaterium, B.stearothermophilus, and/or B. amyloliquefaciens. In some embodiments,the bacterial host cell is a Clostridium species (e.g., C.acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum,C. perfringens, and C. beijerinckii). In some embodiments, the bacterialhost cell is a Corynebacterium species (e.g., C. glutamicum and C.acetoacidophilum). In some embodiments the bacterial host cell is aEscherichia species (e.g., E. coli). In some embodiments, the bacterialhost cell is an Erwinia species (e.g., E. uredovora, E. carotovora, E.ananas, E. herbicola, E. punctata, and E. terreus). In some embodiments,the bacterial host cell is a Pantoea species (e.g., P. citrea, and P.agglomerans). In some embodiments the bacterial host cell is aPseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, andP. sp. D-01 10). In some embodiments, the bacterial host cell is aStreptococcus species (e.g., S. equisimiles, S. pyogenes, and S.uberis). In some embodiments, the bacterial host cell is a Streptomycesspecies (e.g., S. ambofaciens, S. achromogenes, S. avermitilis, S.coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, andS. lividans). In some embodiments, the bacterial host cell is aZymomonas species (e.g., Z. mobilis, and Z. lipolytica).

An exemplary host cell is Escherichia coli W3110. The expression vectorwas created by operatively linking a polynucleotide encoding an improvedPGA into the plasmid pCK110900 operatively linked to the lac promoterunder control of the lad repressor. The expression vector also containedthe P15a origin of replication and the chloramphenicol resistance gene.Cells containing the subject polynucleotide in Escherichia coli W3110were isolated by subjecting the cells to chloramphenicol selection.

Many prokaryotic and eukaryotic strains that find use in the presentinvention are readily available to the public from a number of culturecollections such as American Type Culture Collection (ATCC), DeutscheSammlung von Mikroorganismen and Zellkulturen GmbH (DSM), CentraalbureauVoor Schimmelcultures (CBS), and Agricultural Research Service PatentCulture Collection, Northern Regional Research Center (NRRL).

In some embodiments, host cells are genetically modified to havecharacteristics that improve protein secretion, protein stability and/orother properties desirable for expression and/or secretion of a protein.Genetic modification can be achieved by genetic engineering techniquesand/or classical microbiological techniques (e.g., chemical or UVmutagenesis and subsequent selection). Indeed, in some embodiments,combinations of recombinant modification and classical selectiontechniques are used to produce the host cells. Using recombinanttechnology, nucleic acid molecules can be introduced, deleted, inhibitedor modified, in a manner that results in increased yields of PGAvariant(s) within the host cell and/or in the culture medium. Forexample, knockout of Alpl function results in a cell that is proteasedeficient, and knockout of pyr5 function results in a cell with apyrimidine deficient phenotype. In one genetic engineering approach,homologous recombination is used to induce targeted gene modificationsby specifically targeting a gene in vivo to suppress expression of theencoded protein. In alternative approaches, siRNA, antisense and/orribozyme technology find use in inhibiting gene expression. A variety ofmethods are known in the art for reducing expression of protein incells, including, but not limited to deletion of all or part of the geneencoding the protein and site-specific mutagenesis to disrupt expressionor activity of the gene product. (See e.g., Chaveroche et al., Nucl.Acids Res., 28:22 e97 [2000]; Cho et al., Molec. Plant MicrobeInteract., 19:7-15 [2006]; Maruyama and Kitamoto, Biotechnol Lett.,30:1811-1817 [2008]; Takahashi et al., Mol. Gen. Genom., 272: 344-352[2004]; and You et al., Arch. Micriobiol., 191:615-622 [2009], all ofwhich are incorporated by reference herein). Random mutagenesis,followed by screening for desired mutations also finds use (See e.g.,Combier et al., FEMS Microbiol. Lett., 220:141-8 [2003]; and Firon etal., Eukary. Cell 2:247-55 [2003], both of which are incorporated byreference).

Introduction of a vector or DNA construct into a host cell can beaccomplished using any suitable method known in the art, including butnot limited to calcium phosphate transfection, DEAE-Dextran mediatedtransfection, PEG-mediated transformation, electroporation, or othercommon techniques known in the art.

In some embodiments, the engineered host cells (i.e., “recombinant hostcells”) of the present invention are cultured in conventional nutrientmedia modified as appropriate for activating promoters, selectingtransformants, or amplifying the cellobiohydrolase polynucleotide.Culture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and arewell-known to those skilled in the art. As noted, many standardreferences and texts are available for the culture and production ofmany cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin.

In some embodiments, cells expressing the variant PGA polypeptides ofthe invention are grown under batch or continuous fermentationsconditions. Classical “batch fermentation” is a closed system, whereinthe compositions of the medium is set at the beginning of thefermentation and is not subject to artificial alternations during thefermentation. A variation of the batch system is a “fed-batchfermentation” which also finds use in the present invention. In thisvariation, the substrate is added in increments as the fermentationprogresses. Fed-batch systems are useful when catabolite repression islikely to inhibit the metabolism of the cells and where it is desirableto have limited amounts of substrate in the medium. Batch and fed-batchfermentations are common and well known in the art. “Continuousfermentation” is an open system where a defined fermentation medium isadded continuously to a bioreactor and an equal amount of conditionedmedium is removed simultaneously for processing. Continuous fermentationgenerally maintains the cultures at a constant high density where cellsare primarily in log phase growth. Continuous fermentation systemsstrive to maintain steady state growth conditions. Methods formodulating nutrients and growth factors for continuous fermentationprocesses as well as techniques for maximizing the rate of productformation are well known in the art of industrial microbiology.

In some embodiments of the present invention, cell-freetranscription/translation systems find use in producing variant PGA(s).Several systems are commercially available and the methods arewell-known to those skilled in the art.

The present invention provides methods of making variant PGApolypeptides or biologically active fragments thereof. In someembodiments, the method comprises: providing a host cell transformedwith a polynucleotide encoding an amino acid sequence that comprises atleast about 70% (or at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%) sequenceidentity to SEQ ID NO:2 and comprising at least one mutation as providedherein; culturing the transformed host cell in a culture medium underconditions in which the host cell expresses the encoded variant PGApolypeptide; and optionally recovering or isolating the expressedvariant PGA polypeptide, and/or recovering or isolating the culturemedium containing the expressed variant PGA polypeptide. In someembodiments, the methods further provide optionally lysing thetransformed host cells after expressing the encoded PGA polypeptide andoptionally recovering and/or isolating the expressed variant PGApolypeptide from the cell lysate. The present invention further providesmethods of making a variant PGA polypeptide comprising cultivating ahost cell transformed with a variant PGA polypeptide under conditionssuitable for the production of the variant PGA polypeptide andrecovering the variant PGA polypeptide. Typically, recovery or isolationof the PGA polypeptide is from the host cell culture medium, the hostcell or both, using protein recovery techniques that are well known inthe art, including those described herein. In some embodiments, hostcells are harvested by centrifugation, disrupted by physical or chemicalmeans, and the resulting crude extract retained for furtherpurification. Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including, but not limited tofreeze-thaw cycling, sonication, mechanical disruption, and/or use ofcell lysing agents, as well as many other suitable methods well known tothose skilled in the art.

Engineered PGA enzymes expressed in a host cell can be recovered fromthe cells and or the culture medium using any one or more of the wellknown techniques for protein purification, including, among others,lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli, are commercially available under the trade name CelLytic B™(Sigma-Aldrich). Thus, in some embodiments, the resulting polypeptide isrecovered/isolated and optionally purified by any of a number of methodsknown in the art. For example, in some embodiments, the polypeptide isisolated from the nutrient medium by conventional procedures including,but not limited to, centrifugation, filtration, extraction,spray-drying, evaporation, chromatography (e.g., ion exchange, affinity,hydrophobic interaction, chromatofocusing, and size exclusion), orprecipitation. In some embodiments, protein refolding steps are used, asdesired, in completing the configuration of the mature protein. Inaddition, in some embodiments, high performance liquid chromatography(HPLC) is employed in the final purification steps. For example, in someembodiments, methods known in the art, find use in the present invention(See e.g., Parry et al., Biochem. J., 353:117 [2001]; and Hong et al.,Appl. Microbiol. Biotechnol., 73:1331 [2007], both of which areincorporated herein by reference). Indeed, any suitable purificationmethods known in the art find use in the present invention.

Chromatographic techniques for isolation of the PGA polypeptide include,but are not limited to reverse phase chromatography high performanceliquid chromatography, ion exchange chromatography, gel electrophoresis,and affinity chromatography. Conditions for purifying a particularenzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,are known to those skilled in the art.

In some embodiments, affinity techniques find use in isolating theimproved PGA enzymes. For affinity chromatography purification, anyantibody which specifically binds the PGA polypeptide may be used. Forthe production of antibodies, various host animals, including but notlimited to rabbits, mice, rats, etc., may be immunized by injection withthe PGA. The PGA polypeptide may be attached to a suitable carrier, suchas BSA, by means of a side chain functional group or linkers attached toa side chain functional group. Various adjuvants may be used to increasethe immunological response, depending on the host species, including butnot limited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacillus Calmette Guerin) and Corynebacterium parvum.

In some embodiments, the PGA variants are prepared and used in the formof cells expressing the enzymes, as crude extracts, or as isolated orpurified preparations. In some embodiments, the PGA variants areprepared as lyophilisates, in powder form (e.g., acetone powders), orprepared as enzyme solutions. In some embodiments, the PGA variants arein the form of substantially pure preparations.

In some embodiments, the PGA polypeptides are attached to any suitablesolid substrate. Solid substrates include but are not limited to a solidphase, surface, and/or membrane. Solid supports include, but are notlimited to organic polymers such as polystyrene, polyethylene,polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide,as well as co-polymers and grafts thereof. A solid support can also beinorganic, such as glass, silica, controlled pore glass (CPG), reversephase silica or metal, such as gold or platinum. The configuration ofthe substrate can be in the form of beads, spheres, particles, granules,a gel, a membrane or a surface. Surfaces can be planar, substantiallyplanar, or non-planar. Solid supports can be porous or non-porous, andcan have swelling or non-swelling characteristics. A solid support canbe configured in the form of a well, depression, or other container,vessel, feature, or location. A plurality of supports can be configuredon an array at various locations, addressable for robotic delivery ofreagents, or by detection methods and/or instruments.

In some embodiments, immunological methods are used to purify PGAvariants. In one approach, antibody raised against a variant PGApolypeptide (e.g., against a polypeptide comprising any of SEQ ID NOS:2,4, 6, 8, 10, or 12, and/or an immunogenic fragment thereof) usingconventional methods is immobilized on beads, mixed with cell culturemedia under conditions in which the variant PGA is bound, andprecipitated. In a related approach, immunochromatography finds use.

In some embodiments, the variant PGAs are expressed as a fusion proteinincluding a non-enzyme portion. In some embodiments, the variant PGAsequence is fused to a purification facilitating domain. As used herein,the term “purification facilitating domain” refers to a domain thatmediates purification of the polypeptide to which it is fused. Suitablepurification domains include, but are not limited to metal chelatingpeptides, histidine-tryptophan modules that allow purification onimmobilized metals, a sequence which binds glutathione (e.g., GST), ahemagglutinin (HA) tag (corresponding to an epitope derived from theinfluenza hemagglutinin protein; See e.g., Wilson et al., Cell 37:767[1984]), maltose binding protein sequences, the FLAG epitope utilized inthe FLAGS extension/affinity purification system (e.g., the systemavailable from Immunex Corp), and the like. One expression vectorcontemplated for use in the compositions and methods described hereinprovides for expression of a fusion protein comprising a polypeptide ofthe invention fused to a polyhistidine region separated by anenterokinase cleavage site. The histidine residues facilitatepurification on IMIAC (immobilized metal ion affinity chromatography;See e.g., Porath et al., Prot. Exp. Purif., 3:263-281 [1992]) while theenterokinase cleavage site provides a means for separating the variantPGA polypeptide from the fusion protein. pGEX vectors (Promega) may alsobe used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toligand-agarose beads (e.g., glutathione-agarose in the case ofGST-fusions) followed by elution in the presence of free ligand.

EXPERIMENTAL

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

In the experimental disclosure below, the following abbreviations apply:ppm (parts per million); M (molar); mM (millimolar), uM and μM(micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg(milligrams); ug and μg (micrograms); L and 1 (liter); ml and mL(milliliter); cm (centimeters); mm (millimeters); um and μm(micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s)(hour(s)); U (units); MW (molecular weight); rpm (rotations per minute);° C. (degrees Centigrade); RT (room temperature); CDS (coding sequence);DNA (deoxyribonucleic acid); RNA (ribonucleic acid); TB (Terrific Broth;12 g/L bacto-tryptone, 24 g/L yeast extract, 4 mL/L glycerol, 65 mMpotassium phosphate, pH 7.0, 1 mM MgSO₄); CAM (chloramphenicol); PMBS(polymyxin B sulfate); IPTG (isopropyl thiogalactoside); TFA(trifluoroacetic acid); HPLC (high performance liquid chromatography);FIOPC (fold improvement over positive control); HTP (high throughput);LB (Luria broth); Codexis (Codexis, Inc., Redwood City, Calif.);Sigma-Aldrich (Sigma-Aldrich, St. Louis, Mo.); Millipore (Millipore,Corp., Billerica Mass.); Difco (Difco Laboratories, BD DiagnosticSystems, Detroit, Mich.); Daicel (Daicel, West Chester, Pa.); Genetix(Genetix USA, Inc., Beaverton, Oreg.); Molecular Devices (MolecularDevices, LLC, Sunnyvale, Calif.); Applied Biosystems (AppliedBiosystems, part of Life Technologies, Corp., Grand Island, N.Y.),Agilent (Agilent Technologies, Inc., Santa Clara, Calif.); ThermoScientific (part of Thermo Fisher Scientific, Waltham, Mass.); (Infors;Infors-HT, Bottmingen/Basel, Switzerland); Corning (Corning, Inc., PaloAlto, Calif.); and Bio-Rad (Bio-Rad Laboratories, Hercules, Calif.);Microfluidics (Microfluidics Corp., Newton, Mass., United States ofAmerica).

The following sequences find use in the present invention.

(SEQ ID NO: 1) SEQ ID NO: 1 (PGA WT polynucleotide sequence)ATGAAAAATAGAAATCGTATGATCGTGAACGGTATTGTGACTTCCCTGATCTGTTGTTCTAGCCTGTCAGCGCTGGCGGCAAGCCCGCCAACCGAGGTTAAGATCGTTCGCGATGAATACGGCATGCCGCATATTTACGCCGATGATACCTATCGACTGTTTTACGGCTATGGCTACGTGGTGGCGCAGGATCGCCTGTTCCAGATGGAAATGGCGCGCCGCAGTACTCAGGGGACCGTCTCCGAGGTGCTGGGCAAAGCATTCGTCAGTTTTGATAAAGATATTCGCCAGAACTACTGGCCGGATTCTATTCGCGCGCAGATAGCTTCCCTCTCCGCTGAGGATAAATCCATTCTGCAGGGCTATGCCGATGGCATGAATGCGTGGATCGATAAAGTGAACGCCAGCCCCGATAAGCTGTTACCCCAGCAGTTCTCCACCTTTGGTTTTAAACCCAAGCATTGGGAACCGTTTGATGTGGCGATGATTTTTGTCGGCACCATGGCGAACCGGTTTTCTGACAGCACCAGCGAAATTGATAACCTGGCGCTGCTGACGGCGCTAAAAGATAAATACGGCAAGCAGCAGGGCATGGCGGTCTTTAACCAGCTGAAATGGCTGGTTAATCCTTCCGCGCCAACCACCATTGCGGCGCGGGAAAGCGCCTATCCGCTGAAGTTTGATCTGCAAAACACGCAAACGGCGGCGCTGCTGCCGCGCTACGACCAGCCGGCACCGATGCTCGACCGCCCGGCAAAAGGGACCGATGGCGCGCTGCTGGCGCTGACCGCCGATCAGAACCGGGAAACTATCGCCGCGCAGTTCGCGCAAAGCGGCGCTAACGGCCTGGCTGGCTACCCGACCACTAGCAATATGTGGGTGATTGGCAAAAACAAAGCCCAGGATGCGAAGGCCATTATGGTCAATGGGCCGCAGTTTGGTTGGTATGCGCCGGCGTACACCTACGGTATCGGCCTGCACGGCGCGGGCTATGACGTCACCGGCAATACGCCGTTTGCCTATCCGGGCCTCGTTTTTGGTCACAACGGCACCATTTCATGGGGATCCACCGCCGGTTTTGGTGATGATGTCGATATCTTTGCCGAAAAACTTTCCGCCGAGAAGCCGGGCTATTACCAGCATAACGGCGAGTGGGTGAAGATGTTGAGCCGCAAGGAGACTATTGCGGTCAAAGACGGCCAGCCGGAGACCTTTACCGTTTGGCGCACGCTGCACGGCAACGTCATTAAAACCGATACTGCGACGCAGACCGCCTATGCCAAAGCGCGCGCCTGGGATGGCAAAGAGGTGGCGTCCCTGCTGGCGTGGACGCACCAGATGAAGGCCAAAAACTGGCCGGAGTGGACGCAGCAGGCGGCCAAACAGGCGCTGACCATTAACTGGTACTACGCCGATGTGAACGGCAATATCGGCTATGTGCATACCGGCGCCTATCCGGATCGCCAGCCCGGCCACGACCCGCGTTTGCCGGTTCCCGGCACTGGAAAATGGGACTGGAAAGGGTTGCTGTCGTTTGATTTGAATCCGAAAGTGTATAACCCGCAGTCGGGCTATATCGCCAACTGGAACAACTCGCCGCAAAAAGACTACCCGGCCTCTGATCTGTTCGCGTTCCTGTGGGGCGGTGCGGATCGAGTTACTGAGATCGACACGATCCTCGATAAGCAACCGCGCTTCACCGCCGATCAGGCGTGGGATGTGATCCGCCAAACCAGCCGTCGGGATCTCAACCTGCGGTTGTTCTTACCGGCGCTGAAGGACGCCACCGCGAACCTGGCGGAAAACGATCCGCGCCGCCAACTGGTGGATAAACTGGCGAGCTGGGACGGTGAAAACCTTGTCAACGATGACGGAAAAACCTATCAGCAACCGGGATCGGCGATTCTTAACGCCTGGCTGACCAGCATGCTCAAGCGCACGGTGGTTGCCGCGGTCCCAGCGCCGTTTGGCAAGTGGTACAGCGCCAGTGGCTATGAAACCACCCAGGACGGGCCAACCGGCTCGCTGAACATCAGCGTGGGGGCGAAAATCCTCTACGAAGCTCTGCAGGGTGATAAGTCGCCAATCCCGCAGGCGGTCGATCTGTTTGGCGGGAAACCGCAGCAGGAAGTGATACTGGCGGCGCTGGACGACGCTTGGCAGACGCTGTCAAAACGCTACGGTAACGACGTCACCGGCTGGAAAACCCCTGCCATGGCGCTTACCTTCCGGGCCAATAACTTCTTCGGCGTGCCGCAGGCGGCAGCAAAAGAGGCGCGTCATCAGGCGGAGTACCAGAACCGCGGTACGGAAAACGACATGATTGTCTTCTCACCGACGTCGGGTAACCGCCCGGTTCTTGCCTGGGATGTGGTGGCGCCGGGGCAAAGCGGTTTTATCGCGCCGGATGGCAAAGCCGATAAGCACTATGACGATCAGCTGAAAATGTACGAGAGCTTTGGCCGTAAATCGCTGTGGTTAACGCCTCAGGACGTTGACGAGCACAAAGAGTCTCAGGAAGTGCTGCAGGTACAGCGCTAA (SEQ ID NO: 2)SEQ ID NO: 2 (PGA WT polypeptide sequence)MKNRNRMIVNGIVTSLICCSSLSALAASPPTEVKIVRDEYGMPHIYADDTYRLFYGYGYVVAQDRLFQMEMARRSTQGTVSEVLGKAFVSFDKDIRQNYWPDSIRAQIASLSAEDKSILQGYADGMNAWIDKVNASPDKLLPQQFSTFGFKPKHWEPFDVAMIFVGTMANRFSDSTSEIDNLALLTALKDKYGKQQGMAVFNQLKWLVNPSAPTTIAARESAYPLKFDLQNTQTAALLPRYDQPAPMLDRPAKGTDGALLALTADQNRETIAAQFAQSGANGLAGYPTTSNMWVIGKNKAQDAKAIMVNGPQFGWYAPAYTYGIGLHGAGYDVTGNTPFAYPGLVFGHNGTISWGSTAGFGDDVDIFAEKLSAEKPGYYQHNGEWVKMLSRKETIAVKDGQPETFTVWRTLHGNVIKTDTATQTAYAKARAWDGKEVASLLAWTHQMKAKNWPEWTQQAAKQALTINWYYADVNGNIGYVHTGAYPDRQPGHDPRLPVPGTGKWDWKGLLSFDLNPKVYNPQSGYIANVVNNSPQKDYPASDLFAFLWGGADRVTEIDTILDKQPRFTADQAWDVIRQTSRRDLNLRLFLPALKDATANLAENDPRRQLVDKLASWDGENLVNDDGKTYQQPGSAILNAWLTSMLKRTVVAAVPAPFGKWYSASGYETTQDGPTGSLNISVGAKILYEALQGDKSPIPQAVDLFGGKPQQEVILAALDDAWQTLSKRYGNDVTGWKTPAMALTFRANNFFGVPQAAAKEARHQAEYQNRGTENDMIVFSPTSGNRPVLAWDVVAPGQSGFIAPDGKADKHYDDQLKMYESFGRKSLWLTPQDVDEHKESQEVLQVQR (SEQ ID NO: 3)SEQ ID NO: 3 (PGA Variant 1 polynucleotide sequence)AGCAATATGTGGGTGATTGGCAAAAACAAAGCCCAGGATGCGAAGGCCATTATGGTCAATGGGCCGCAGTTTGGTTGGTATGTGCCGGCGTACACCTACGGTATCGGCCTGCACGGCGCGGGCTATGACGTCACCGGCAATACGCCGTTTGCCTATCCGGGCCTCGTTTTTGGTCACAACGGCACCATTTCATGGGGATCCACCGCCGGTGGTGGTGATGATGTCGATATCTTTGCCGAAAAACTTTCCGCCGAGAAGCCGGGCTATTACCAGCATAACGGCGAGTGGGTGAAGATGTTGAGCCGCAAGGAGACTATTGCGGTCAAAGACGGCCAGCCGGAGACCTTTACCGTTTGGCGCACGCTGCACGGCAACGTCATTAAAACCGATACTGCGACGCAGACCGCCTATGCCAAAGCGCGCGCCTGGGATGGCAAAGAGGTGGCGTCCCTGCTGGCGTGGACGCACCAGATGAAGGCCAAAAACTGGCCGGAGTGGACGCAGCAGGCGGCCAAACAGGCGCTGACCATTAACTGGTACTACGCCGATGTGAACGGCAATATCGGCTATGTGCATACCGGCGCCTATCCGGATCGCCAGCCCGGCCACGACCCGCGTTTGCCGGTTCCCGGCACTGGAAAATGGGACTGGAAAGGGTTGCTGTCGTTTGATTTGAATCCGAAAGTGTATAACCCGCAGTCGGGCTATATCGCCAACTGGAACAACTCGCCGCAAAAAGACTACCCGGCCTCTGATCTGTTCGCGTTCCTGTGGGGCGGTGCGGATCGAGTTACTGAGATCGACACGATCCTCGATAAGCAACCGCGCTTCACCGCCGATCAGGCGTGGGATGTGATCCGCCAAACCAGCCGTCGGGATCTCAACCTGCGGTTGTTCTTACCGGCGCTGAAGGACGCCACCGCGAACCTGGCGGAAAACGATCCGCGCCGCCAACTGGTGGATAAACTGGCGAGCTGGGACGGCGAAAACCTTGTCAACGATGACGGAAAAACCTATCAGCAACCGGGATCGGCGATTCTTAACGCCTGGCTGACCAGCATGCTCAAGCGCACGGTGGTTGCCGCGGTCCCAGCGCCGTTTGGTAAGTGGTACAGCGCCAGTGGCTATGAAACCACCCAGGACGGGCCAACCGGCTCGCTGAACATCAGCGTGGGGGCGAAAATCCTCTACGAAGCTCTGCAGGGTGATAAGTCGCCAATCCCGCAGGCGGTCGATCTGTTTGGCGGGAAACCGCAGCAGGAAGTAATACTGGCGGCGCTGGACGACGCTTGGCAGACGCTGTCAAAACGCTACGGTAACGACGTCACCGGCTGGAAAACCCCTGCCATGGCGCTTACCTTCCGGGCCAATAACTTCTTCGGCGTGCCGCAGGCGGCAGCAAAAGAGGCGCGTCATCAGGCGGAGTACCAGAACCGCGGTACGGAAAACGACATGATTGTCTTCTCACCGACGTCGGGTAACCGCCCGGTTCTTGCCTGGGATGTGGTGGCGCCGGGGCAAAGCGGTTTTATCGCGCCGGATGGCAAAGCCGATAAGCACTATGACGATCAGCTGAAAATGTACGAGAGCTTTGGCCGTAAATCGCTGTGGTTAACGCCTCAGGACGTTGACGAGCACCAAGAGTCTCAGGAAGTGCTGCAGGTACAGTTGGATCAGACCGAGGTTAAGATCGTTCGCGATGAATACGGCATGCCGCATATTTACGCCGATGATACCTATCGACTGTTTTACGGCTATGGCTACGTGGTGGCGCAGGATCGCCTGTTCCAGATGGAAATGGCGCGCCGCAGTACTCAGGGGACCGTCTCCGAGGTGCTGGGCAAAGCATTCGTCAGTTTTGATAAAGATATTCGCCAGAACTACTGGCCGGATTCTATTCGCGCGCAGATAGCTTCCCTCTCCGCTGAGGATAAATCCATTCTGCAGGGCTATGCCGATGGCATGAATGCGTGGATCGATAAAGTGAACGCCAGCCCCGATAAGCTGTTACCCCAGCAGTTCTCCACCTTTGGTTTTAAACCCAAGCATTGGGAACCGTTTGATGTGGCGATGATTTTTGTCGGCACCATGGCGAACCGTTTCTCTGACAGCACCAGCGAAATTGATAACCTGGCGCTGCTGACGGCGCTAAAAGACAAATACGGCAAGCAGCAGGGCATGGCGGTCTTTAACCAGCTGAAATGGCTGGTTAATCCTTCCGCGCCAACCACCATTGCGGCGCGGGAAAGCGCCTATCCGCTGAAGTTTGATCTGCAAAACACGCAAACGGCGTAA (SEQ ID NO: 4)SEQ ID NO: 4 (PGA Variant 1 polypeptide sequence)SNMWVIGKNKAQDAKAIMVNGPQFGWYVPAYTYGIGLHGAGYDVTGNTPFAYPGLVFGHNGTISWGSTAGGGDDVDIFAEKLSAEKPGYYQHNGEWVKMLSRKETIAVKDGQPETFTVWRTLHGNVIKTDTATQTAYAKARAWDGKEVASLLAWTHQMKAKNVVPEWTQQAAKQALTINWYYADVNGNIGYVHTGAYPDRQPGHDPRLPVPGTGKWDWKGLLSFDLNPKVYNPQSGYIANWNNSPQKDYPASDLFAFLWGGADRVTEIDTILDKQPRFTADQAWDVIRQTSRRDLNLRLFLPALKDATANLAENDPRRQLVDKLASWDGENLVNDDGKTYQQPGSAILNAWLTSMLKRTVVAAVPAPFGKWYSASGYETTQDGPTGSLNISVGAKILYEALQGDKSPIPQAVDLFGGKPQQEVILAALDDAWQTLSKRYGNDVTGWKTPAMALTFRANNFFGVPQAAAKEARHQAEYQNRGTENDMIVFSPTSGNRPVLAWDVVAPGQSGFIAPDGKADKHYDDQLKMYESFGRKSLWLTPQDVDEHQESQEVLQVQLDQTEVKIVRDEYGMPHIYADDTYRLFYGYGYVVAQDRLFQMEMARRSTQGTVSEVLGKAFVSFDKDIRQNYWPDSIRAQIASLSAEDKSILQGYADGMNAWIDKVNASPDKLLPQQFSTFGFKPKHWEPFDVAMIFVGTMANRFSDSTSEIDNLALLTALKDKYGKQQGMAVFNQLKWLVNPSAPTTIAARESAYPLKFDLQNTQTA (SEQ ID NO: 5)SEQ ID NO: 5 (PGA Variant 6 polynucleotide sequence)AGCAATATGTGGGTGATTGGCAAAAACAAAGCCCAGGATGCGAAGGCCATTATGGTCAATGGGCCGCAGTTTGGTTGGTATGTGCCGGCGTATACCTACGGTATCGGCCTGCACGGCGCGGGCTATGACGTCACCGGCAATACGCCGTTTGCCTATCCGGGCCTCGTTTTTGGTCACAACGGCACCATTTCATGGGGATCCACCGCCGGTGGTGGTGATGATGTCGATATCTTTGCCGAAAAACTTTCCGCCGAGAAGCCGGGCTATTACCAGCATAACGGCGAGTGGGTGAAGATGTTGAGCCGCAAGGAGACTATTGCGGTCAAAGACGGCCAGCCGGAGACCTTTACCGTTTGGCGCACGCTGCACGGCAACGTCATTAAAACCGATACTGCGACGCAGACCGCCTATGCCAAAGCGCGCGCCTGGGATGGCAAAGAGGTGGCGTCCCTGCTGGCGTGGACGCACCAGATGAAGGCCAAAAACTGGCCGGAGTGGACGCAGCAGGCGGCCAAACAGGCGCTGACCATCAACTGGTACTACGCCGATGTGAACGGCAATATCGGCTATGTGCATACCGGCGCCTATCCGGATCGCCAGCCCGGCCACGACCCGCGTTTGCCGGTTCCCGGCACTGGAAAATGGGACTGGAAAGGGTTGCTGTCGTTTGATTTGAATCCGAAAGTGTATAACCCGCAGTCGGGCTATATCGCCAACTGGAACAACTCGCCGCAAAAAGACTACCCGGCCTCTGATCTGTTCGCGTTCCTGTGGGGCGGTGCGGATCGAGCGACTGAGATCGACACGATCCTCGATAAGCAACCGCGCTTCACCGCCGATCAGGCGTGGGATGTGATCCGCCAAACCAGCCGTCGGGATCTCAACCTGCGGTTGTTCTTACCGGCGCTGAAGGACGCCACCGCCAACCTGGCGGAAAACGATCCGCGCCGCCAACTGGTGGATAAACTGGCGAGCTGGGACGGCGAAAACCTTGTCAACGATGACGGAAAAACCTATCAGCAACCGGGATCGGCGATTCTTAACGCCTGGCTGACCAGCATGCTCAAGCGCACGGTGGTTGCCGCGGTCCCAGCGCCGTTTGGTAAGTGGTACAGCGCCAGTGGCTATGAAACCACCCAGGACGGGCCAACCGGCTCGCTGAACATCAGCGTGGGGGCGAAAATCCTCTACGAAGCTCTGCAGGGTGATAAGTCGCCAATCCCGCAGGCGGTCGATCTGTTTGGCGGGAAACCGCAGCAGGAAGTAATACTGGCGGCGCTGGACGACGCTTGGCAGACGCTGTCAAAACGCTACGGTAACGACGTCACCGGCTGGAAAACCCCTGCCATGGCGCTTACCTTCCGGGCCAATAACTTCTTCGGCGTGCCGCAGGCGGCAGCAAAAGAGGCGCGTCATCAGGCGGAGTACCAGAACCGCGGTACGGAAAACAACATGATTGTCTTCTCACCGACGTCGGGTAACCGCCCGGTTCTTGCCTGGGATGTGGTGGCGCCGGGGCAAAGCGGTTTTATCGCGCCGGATGGCAAAGCCGATAAGCACTATGACGATCAGCTGAAAATGTACGAGAGCTTTGGCCGTAAATCGCTGTGGTTAACGCCTCAGGACGTTGACGAGCACAAAGAGTCTCAGGAAGTGCTGCAGGTACAGTTGGATCAGACCGAGGTTAAGATCGTTCGCGATGAATACGGCATGCCGCATATTTACGCCGATGATACCTATCGACTGTTTTACGGCTATGGCTACGTGGTGGCGCAGGATCGCCTGTTCCAGATGGAAATGGCGCGCCGCAGTACTCAGGGGACCGTCTCCGAGGTGCTGGGCAAAGCATTCGTCAGTTTTGATAAAGATATTCGCCAGAACTACTGGCCGGATTCTATTCGCGCGCAGATAGCTTCCCTCTCCGCTGAGGATAAATCCATTCTGCAGGGCTATGCCGATGGCATGAATGCGTGGATCGATAAAGTGAACGCCAGCCCCGATAAGCTGTTACCCCAGCAGTTCTCCACCTTTGGTTTTAAACCCAAGCATTGGGAACCGTTTGATGTGGCGATGATTTTTGTCGGCACCATGGCGAACCGTTTTTCTGACAGCACCAGCGAAATTGATAACCTGGCGCTGCTGACGGCGCTAAAAGACAAATACGGCAAGCAGCAGGGCATGGCGGTCTTTAACCAGCTGAAATGGCTGGTTAATCCTTCCGCGCCAACCACCATTGCGGCGCGGGAAAGCGCCTATCCGCTGAAGTTTGATCTGCAAAACACGCAAACGGCGTAA (SEQ ID NO: 6)SEQ ID NO: 6 (PGA Variant 6 polypeptide sequence)SNMWVIGKNKAQDAKAIMVNGPQFGWYVPAYTYGIGLHGAGYDVTGNTPFAYPGLVFGHNGTISWGSTAGGGDDVDIFAEKLSAEKPGYYQHNGEWVKMLSRKETIAVKDGQPETFTVWRTLHGNVIKTDTATQTAYAKARAWDGKEVASLLAWTHQMKAKNVVPEWTQQAAKQALTINWYYADVNGNIGYVHTGAYPDRQPGHDPRLPVPGTGKWDWKGLLSFDLNPKVYNPQSGYIANWNNSPQKDYPASDLFAFLWGGADRATEIDTILDKQPRFTADQAWDVIRQTSRRDLNLRLFLPALKDATANLAENDPRRQLVDKLASWDGENLVNDDGKTYQQPGSAILNAWLTSMLKRTVVAAVPAPFGKWYSASGYETTQDGPTGSLNISVGAKILYEALQGDKSPIPQAVDLFGGKPQQEVILAALDDAWQTLSKRYGNDVTGWKTPAMALTFRANNFFGVPQAAAKEARHQAEYQNRGTENNMIVFSPTSGNRPVLAWDVVAPGQSGFIAPDGKADKHYDDQLKMYESFGRKSLWLTPQDVDEHKESQEVLQVQLDQTEVKIVRDEYGMPHIYADDTYRLFYGYGYVVAQDRLFQMEMARRSTQGTVSEVLGKAFVSFDKDIRQNYWPDSIRAQIASLSAEDKSILQGYADGMNAWIDKVNASPDKLLPQQFSTFGFKPKHWEPFDVAMIFVGTMANRFSDSTSEIDNLALLTALKDKYGKQQGMAVFNQLKWLVNPSAPTTIAARESAYPLKFDLQNTQTA (SEQ ID NO: 7)SEQ ID NO: 7 (PGA Variant 53 polynucleotide sequence)AGCAATATGTGGGTGATTGGCAAAAACAAAGCCCAGGATGCGAAGGCCATTATGGTCAATGGGCCGCAGTTTGGTTGGTTTAATCCGGCGTACACCTACGGTATCGGCCTGCACGGCGCGGGCTATGACGTCACCGGCAATACGCCGTTTGCCTATCCGGGCCTCCTGTTTGGTCACAACGGCACCATTTCATGGGGATCCACCGCCGGTGGTGGTGATGATGTCGATATCTTTGCCGAAAAACTTTCCGCCGAGAAGCCGGGCTATTACCAGCATAACGGCGAGTGGGTGAAGATGTTGAGCCGCAAGGAGACTATTGCGGTCAAAGACGGCCAGCCGGAGACCTTTACCGTTTGGCGCACGCTGCACGGCAACGTCATTAAAACCGATACTGCGACGCAGACCGCCTATGCCAAAGCGCGCGCCTGGGATGGCAAAGAGGTGGCGTCCCTGCTGGCGTGGACGCACCAGATGAAGGCCAAAAACTGGCCGGAGTGGACGCAGCAGGCGGCCAAACAGGCGCTGACCATTAACTGGTACTACGCCGATGTGAACGGCAATATCGGCTATGTGCATACCGGCGCCTATCCGGATCGCCAGCCCGGCCACGACCCGCGTTTGCCGGTTCCCGGCACTGGAAAATGGGACTGGAAAGGGTTGCTGTCGTTTGATTTGAATCCGAAAGTGTATAACCCGCAGTCGGGCTATATCGCCAACTGGAACAACTCGCCGCAAAAAGACTACCCGGCCTCTGATCTGTTCGCGTTCCTGTGGGGCGGTGCGGATCGAGTTACTGAGATCGACACGATCCTCGATAAGCAACCGCGCTTCACCGCCGATCAGGCGTGGGATGTGATCCGCCAAACCAGCCGTCGGGATCTCAACCTGCGGTTGTTCTTACCGGCGCTGAAGGACGCCACCGCGAACCTGGCGGAAAACGATCCGCGCCGCCAACTGGTGGATAAACTGGCGAGCTGGGACGGCGAAAACCTTGTCAACGATGACGGAAAAACCTATCAGCAACCGGGATCGGCGATTCTTAACGCCTGGCTGACCAGCATGCTCAAGCGCACGGTGGTTGCCGCGGTCCCAGCGCCGTTTGGTAAGTGGTACAGCGCCAGTGGCTATGAAACCACCCAGGACGGGCCAACCGGCTCGCTGAACATCAGCGTGGGGGCGAAAATCCTCTACGAAGCTCTGCAGGGTGATAAGTCGCCAATCCCGCAGGCGGTCGATCTGTTTGGCGGGAAACCGCAGCAGGAAGTAATACTGGCGGCGCTGGACGACGCTTGGCAGACGCTGTCAAAACGCTACGGTAACGACGTCACCGGCTGGAAAACCCCTGCCATGGCGCTTACCTTCCGGGCCAATAACTTCTTCGGCGTGCCGCAGGCGGCAGCAAAAGAGGCGCGTCATCAGGCGGAGTACCAGAACCGCGGTACGGAAAACGACATGATTGTCTTCTCACCGACGTCGGGTAACCGCCCGGTTCTTGCCTGGGATGTGGTGGCGCCGGGGCAAAGCGGTTTTATCGCGCCGGATGGCAAAGCCGATAAGCACTATGACGATCAGCTGAAAATGTACGAGAGCTTTGGCCGTAAATCGCTGTGGTTAACGCCTCAGGACGTTGACGAGCACCAAGAGTCTCAGGAAGTGCTGCAGGTACAGTTGGATCAGACCGAGGTTAAGATCGTTCGCGATGAATACGGCATGCCGCATATTTACGCCGATGATACCTATCGACTGTTTTACGGCTATGGCTACGTGGTGGCGCAGGATCGCCTGTTCCAGATGGAAATGGCGCGCCGCAGTACTCAGGGGACCGTCTCCGAGGTGCTGGGCAAAGCTTTCGTTTCTTTTGATAAAGATATTCGCCAGAACTACTGGCCGGATTCTATTCGCGCGCAGATAGCTTCCCTCTCCGCTGAGGATAAATCCATTCTGCAGGGCTATGCCGATGGCATGAATGCGTGGATCGATAAAGTGAACGCCAGCCCCGATAAGCTGTTACCCCAGCAGTTCTCCACCTTTGGTTTTAAACCCAAGCATTGGGAACCGTTTGATGTGGCGATGATTTTTGTCGGCACCATGGCGAACCGTTTCTCTGACAGCACCAGCGAAATTGATAACCTGGCGCTGCTGACGGCGCTAAAAGACAAATACGGCAAGCAGCAGGGCATGGCGGTCTTTAACCAGCTGAAATGGCTGGTTAATCCTTCCGCGCCAACCACCATTGCGGCGCGGGAAAGCGCCTATCCGCTGAAGTTTGATCTGCAAAACACGCAAACGGCGTAA (SEQ ID NO: 8)SEQ ID NO: 8 (PGA Variant 53 polypeptide sequence)SNMWVIGKNKAQDAKAIMVNGPQFGWFNPAYTYGIGLHGAGYDVTGNTPFAYPGLLFGHNGTISWGSTAGGGDDVDIFAEKLSAEKPGYYQHNGEWVKMLSRKETIAVKDGQPETFTVWRTLHGNVIKTDTATQTAYAKARAWDGKEVASLLAWTHQMKAKNWPEWTQQAAKQALTINWYYADVNGNIGYVHTGAYPDRQPGHDPRLPVPGTGKWDWKGLLSFDLNPKVYNPQSGYIANWNNSPQKDYPASDLFAFLWGGADRVTEIDTILDKQPRFTADQAWDVIRQTSRRDLNLRLFLPALKDATANLAENDPRRQLVDKLASWDGENLVNDDGKTYQQPGSAILNAWLTSMLKRTVVAAVPAPFGKWYSASGYETTQDGPTGSLNISVGAKILYEALQGDKSPIPQAVDLFGGKPQQEVILAALDDAWQTLSKRYGNDVTGWKTPAMALTFRANNFFGVPQAAAKEARHQAEYQNRGTENDMIVFSPTSGNRPVLAWDVVAPGQSGFIAPDGKADKHYDDQLKMYESFGRKSLWLTPQDVDEHQESQEVLQVQLDQTEVKIVRDEYGMPHIYADDTYRLFYGYGYVVAQDRLFQMEMARRSTQGTVSEVLGKAFVSFDKDIRQNYWPDSIRAQIASLSAEDKSILQGYADGMNAWIDKVNASPDKLLPQQFSTFGFKPKHWEPFDVAMIFVGTMANRFSDSTSEIDNLALLTALKDKYGKQQGMAVFNQLKWLVNPSAPTTIAARESAYPLKFDLQNTQTA (SEQ ID NO: 9)SEQ ID NO: 9 (PGA Variant 261 polynucleotide sequence)AGCAATATGTGGGTGATTGGCAAAAACAAAGCCCAGGATGCGAAGGCCATTATGGTCAATGGGCCGCAGTTTGGTTGGTATAATCCGGCGTATACCTACGGTATCGGCCTGCACGGCGCGGGCTATGACGTCACCGGCAATACGCCGTTTGCCTATCCGGGCCTCCTTTTTGGTCACAACGGCACCATTTCATGGGGATCCACCGCCGGTGCCGGTGATGTCGTCGATATCTTTGCCGAAAAACTTTCCGCCGAGAAGCCGGGCTATTACCAGCATAACGGCGAGTGGGTGAAGATGTTGAGCCGCAAGGAGACTATTGCGGTCAAAGACGGCCAGCCGGAGACCTTTACCGTTTGGCGCACGCTGCACGGCAACGTCATTAAAACCGATACTGCGACGCAGACCGCCTATGCCAAAGCGCGCGCCTGGGATGGCAAAGAGGTGGCGTCCCTGCTGGCGTGGACGCACCAGATGAAGGCCAAAAACTGGCCGGAGTGGACGCAGCAGGCGGCCAAACAGGCGCTGACCATCAACTGGTACTACGCCGATGTGAACGGCAATATCGGCTATGTGCATACCGGCGCCTATCCGGATCGCCAGCCCGGCCACGACCCGCGTTTGCCGGTTCCCGGCACTGGAAAATGGGACTGGAAAGGGTTGCTGTCGTTTGATTTGAATCCGAAAGTGTATAACCCGCAGTCGGGCTATATCGCCAACTGGAACAACTCGCCGCAAAAAGACTACCCGGCCTCTGATCTGTTCGCGTTCCTGTGGGGCGGTGCGGATCGAGCGACTGAGATCGACACGATCCTCGATAAGCAACCGCGCTTCACCGCCGATCAGGCGTGGGATGTGATCCGCCAAACCAGCCGTCGGGATCTCAACCTGCGGTTGTTCTTACCGGCGCTGAAGGACGCCACCGCCAACCTGGCGGAAAACGATCCGCGCCGCCAACTGGTGGATAAACTGGCGAGCTGGGACGGCGAAAACCTTGTCAACGATGACGGAAAAACCTATCAGCAACCGGGATCGGCGATTCTTAACGCCTGGCTGACCAGCATGCTCAAGCGCACGGTGGTTGCCGCGGTCCCAGCGCCGTTTGGTAAGTGGTACAGCGCCAGTGGCTATGAAACCACCCAGGACGGGCCAACCGGCTCGCTGAACATCAGCGTGGGGGCGAAAATCCTCTACGAAGCTCTGCAGGGTGATAAGTCGCCAATCCCGCAGGCGGTCGATCTGTTTGGCGGGAAACCGCAGCAGGAAGTAATACTGGCGGCGCTGGACGACGCTTGGCAGACGCTGTCAAAACGCTACGGTAACGACGTCACCGGCTGGAAAACCCCTGCCATGGCGCTTACCTTCCGGGCCAATAACTTCTTCGGCGTGCCGCAGGCGGCAGCAAAAGAGGCGCGTCATCAGGCGGAGTACCAGAACCGCGGTACGGAAAACAACATGATTGTCTTCTCACCGACGTCGGGTAACCGCCCGGTTCTTGCCTGGGATGTGGTGGCGCCGGGGCAAAGCGGTTTTATCGCGCCGGATGGCAAAGCCGATAAGCACTATGACGATCAGCTGAAAATGTACGAGAGCTTTGGCCGTAAATCGCTGTGGTTAACGCCTCAGGACGTTGACGAGCACAAAGAGTCTCAGGAAGTGCTGCAGGTACAGTTGGATCAGACCGAGGTTAAGATCGTTCGCGATGAATACGGCATGCCGCATATTTACGCCGATGATACCTATCGACTGTTTTACGGCTATGGCTACGTGGTGGCGCAGGATCGCCTGTTCCAGATGGAAATGGCGCGCCGCAGTACTCAGGGGACCGTCTCCGAGGTGCTGGGCAAAGCATTCGTTTCATTTGATAAAGATATTCGCCAGAACTACTGGCCGGATTCTATTCGCGCGCAGATAGCTTCCCTCTCCGCTGAGGATAAATCCATTCTGCAGGGCTATGCCGATGGCATGAATGCGTGGATCGATAAAGTGAACGCCAGCCCCGATAAGCTGTTACCCCAGCAGTTCTCCACCTTTGGTTTTAAACCCAAGCATTGGGAACCGTTTGATGTGGCGATGATTTTTGTCGGCACCATGGCGAACCGTTTTTCTGACAGCACCAGCGAAATTGATAACCTGGCGCTGCTGACGGCGCTAAAAGACAAATACGGCAAGCAGCAGGGCATGGCGGTCTTTAACCAGCTGAAATGGCTGGTTAATCCTTCCGCGCCAACCACCATTGCGGCGCGGGAAAGCGCCTATCCGCTGAAGTTTGATCTGCAAAACACGCAAACGGCGTAA (SEQ ID NO: 10)SEQ ID NO: 10 (PGA Variant 261 polypeptide sequence)SNMWVIGKNKAQDAKAIMVNGPQFGWYNPAYTYGIGLHGAGYDVTGNTPFAYPGLLFGHNGTISWGSTAGAGDVVDIFAEKLSAEKPGYYQHNGEWVKMLSRKETIAVKDGQPETFTVWRTLHGNVIKTDTATQTAYAKARAWDGKEVASLLAWTHQMKAKNWPEWTQQAAKQALTINWYYADVNGNIGYVHTGAYPDRQPGHDPRLPVPGTGKWDWKGLLSFDLNPKVYNPQSGYIANWNNSPQKDYPASDLFAFLWGGADRATEIDTILDKQPRFTADQAWDVIRQTSRRDLNLRLFLPALKDATANLAENDPRRQLVDKLASWDGENLVNDDGKTYQQPGSAILNAWLTSMLKRTVVAAVPAPFGKWYSASGYETTQDGPTGSLNISVGAKILYEALQGDKSPIPQAVDLFGGKPQQEVILAALDDAWQTLSKRYGNDVTGWKTPAMALTFRANNFFGVPQAAAKEARHQAEYQNRGTENNMIVFSPTSGNRPVLAWDVVAPGQSGFIAPDGKADKHYDDQLKMYESFGRKSLWLTPQDVDEHKESQEVLQVQLDQTEVKIVRDEYGMPHIYADDTYRLFYGYGYVVAQDRLFQMEMARRSTQGTVSEVLGKAFVSFDKDIRQNYWPDSIRAQIASLSAEDKSILQGYADGMNAWIDKVNASPDKLLPQQFSTFGFKPKHWEPFDVAMIFVGTMANRFSDSTSEIDNLALLTALKDKYGKQQGMAVFNQLKWLVNPSAPTTIAARESAYPLKFDLQNTQTA (SEQ ID NO: 11)SEQ ID NO: 11 (PGA Variant 258 polynucleotide sequence)AGCAATATGTGGGTGATTGGCAAAAACAAAGCCCAGGATGCGAAGGCCATTATGGTCAATGGGCCGCAGTTTGGTTGGTATAATCCGGCGTATACCTACGGTATCGGCCTGCACGGCGCGGGCTATGACGTCACCGGCAATACGCCGTTTGCCTATCCGGGCCTCCTTTTTGGTCACAACGGCACCATTTCATGGGGATCCACCGCCGGTGCCGGTGATAGCGTCGATATCTTTGCCGAAAAACTTTCCGCCGAGAAGCCGGGCTATTACCAGCATAACGGCGAGTGGGTGAAGATGTTGAGCCGCAAGGAGACTATTGCGGTCAAAGACGGCCAGCCGGAGACCTTTACCGTTTGGCGCACGCTGCACGGCAACGTCATTAAAACCGATACTGCGACGCAGACCGCCTATGCCAAAGCGCGCGCCTGGGATGGCAAAGAGGTGGCGTCCCTGCTGGCGTGGACGCACCAGATGAAGGCCAAAAACTGGCCGGAGTGGACGCAGCAGGCGGCCAAACAGGCGCTGACCATCAACTGGTACTACGCCGATGTGAACGGCAATATCGGCTATGTGCATACCGGCGCCTATCCGGATCGCCAGCCCGGCCACGACCCGCGTTTGCCGGTTCCCGGCACTGGAAAATGGGACTGGAAAGGGTTGCTGTCGTTTGATTTGAATCCGAAAGTGTATAACCCGCAGTCGGGCTATATCGCCAACTGGAACAACTCGCCGCAAAAAGACTACCCGGCCTCTGATCTGTTCGCGTTCCTGTGGGGCGGTGCGGATCGAGCGACTGAGATCGACACGATCCTCGATAAGCAACCGCGCTTCACCGCCGATCAGGCGTGGGATGTGATCCGCCAAACCAGCCGTCGGGATCTCAACCTGCGGTTGTTCTTACCGGCGCTGAAGGACGCCACCGCCAACCTGGCGGAAAACGATCCGCGCCGCCAACTGGTGGATAAACTGGCGAGCTGGGACGGCGAAAACCTTGTCAACGATGACGGAAAAACCTATCAGCAACCGGGATCGGCGATTCTTAACGCCTGGCTGACCAGCATGCTCAAGCGCACGGTGGTTGCCGCGGTCCCAGCGCCGTTTGGTAAGTGGTACAGCGCCAGTGGCTATGAAACCACCCAGGACGGGCCAACCGGCTCGCTGAACATCAGCGTGGGGGCGAAAATCCTCTACGAAGCTCTGCAGGGTGATAAGTCGCCAATCCCGCAGGCGGTCGATCTGTTTGGCGGGAAACCGCAGCAGGAAGTAATACTGGCGGCGCTGGACGACGCTTGGCAGACGCTGTCAAAACGCTACGGTAACGACGTCACCGGCTGGAAAACCCCTGCCATGGCGCTTACCTTCCGGGCCAATAACTTCTTCGGCGTGCCGCAGGCGGCAGCAAAAGAGGCGCGTCATCAGGCGGAGTACCAGAACCGCGGTACGGAAAACAACATGATTGTCTTCTCACCGACGTCGGGTAACCGCCCGGTTCTTGCCTGGGATGTGGTGGCGCCGGGGCAAAGCGGTTTTATCGCGCCGGATGGCAAAGCCGATAAGCACTATGACGATCAGCTGAAAATGTACGAGAGCTTTGGCCGTAAATCGCTGTGGTTAACGCCTCAGGACGTTGACGAGCACAAAGAGTCTCAGGAAGTGCTGCAGGTACAGTTGGATCAGACCGAGGTTAAGATCGTTCGCGATGAATACGGCATGCCGCATATTTACGCCGATGATACCTATCGACTGTTTTACGGCTATGGCTACGTGGTGGCGCAGGATCGCCTGTTCCAGATGGAAATGGCGCGCCGCAGTACTCAGGGGACCGTCTCCGAGGTGCTGGGCAAAGCATTCGTTAAGTTTGATAAAGATATTCGCCAGAACTACTGGCCGGATTCTATTCGCGCGCAGATAGCTTCCCTCTCCGCTGAGGATAAATCCATTCTGCAGGGCTATGCCGATGGCATGAATGCGTGGATCGATAAAGTGAACGCCAGCCCCGATAAGCTGTTACCCCAGCAGTTCTCCACCTTTGGTTTTAAACCCAAGCATTGGGAACCGTTTGATGTGGCGATGATTTTTGTCGGCACCATGGCGAACCGTTTTTCTGACAGCACCAGCGAAATTGATAACCTGGCGCTGCTGACGGCGCTAAAAGACAAATACGGCAAGCAGCAGGGCATGGCGGTCTTTAACCAGCTGAAATGGCTGGTTAATCCTTCCGCGCCAACCACCATTGCGGCGCGGGAAAGCGCCTATCCGCTGAAGTTTGATCTGCAAAACACGCAAACGGCGTAA (SEQ ID NO: 12)SEQ ID NO: 12 (PGA Variant 258 polypeptide sequence)SNMWVIGKNKAQDAKAIMVNGPQFGWYNPAYTYGIGLHGAGYDVTGNTPFAYPGLLFGHNGTISWGSTAGAGDSVDIFAEKLSAEKPGYYQHNGEWVKMLSRKETIAVKDGQPETFTVWRTLHGNVIKTDTATQTAYAKARAWDGKEVASLLAWTHQMKAKNWPEWTQQAAKQALTINWYYADVNGNIGYVHTGAYPDRQPGHDPRLPVPGTGKWDWKGLLSFDLNPKVYNPQSGYIANWNNSPQKDYPASDLFAFLWGGADRATEIDTILDKQPRFTADQAWDVIRQTSRRDLNLRLFLPALKDATANLAENDPRRQLVDKLASWDGENLVNDDGKTYQQPGSAILNAWLTSMLKRTVVAAVPAPFGKWYSASGYETTQDGPTGSLNISVGAKILYEALQGDKSPIPQAVDLFGGKPQQEVILAALDDAWQTLSKRYGNDVTGWKTPAMALTFRANNFFGVPQAAAKEARHQAEYQNRGTENNMIVFSPTSGNRPVLAWDVVAPGQSGFIAPDGKADKHYDDQLKMYESFGRKSLWLTPQDVDEHKESQEVLQVQLDQTEVKIVRDEYGMPHIYADDTYRLFYGYGYVVAQDRLFQMEMARRSTQGTVSEVLGKAFVKFDKDIRQNYWPDSIRAQIASLSAEDKSILQGYADGMNAWIDKVNASPDKLLPQQFSTFGFKPKHWEPFDVAMIFVGTMANRFSDSTSEIDNLALLTALKDKYGKQQGMAVFNQLKWLVNPSAPTTIAARESAYPLKFDLQNTQTA

Example 1 E. coli Expression Hosts Containing Recombinant PGA Genes

The initial PGA enzymes used to produce the variants of the presentinvention were obtained from the Codex® Acylase Panel (Codexis). The PGApanel plate comprises a collection of engineered PGA polypeptides thathave improved properties, as compared to the wild-type Kluyveracitrophila PGA. The wild type PGA gene is a heterodimer consisting ofalpha subunit (23.8 KDa) and beta subunit (62.2 KDa) linked by 54aaspacer region. Due to the presence of spacer region, an autoprocessingstep is required to form the active protein. The wild-type gene wasmodified to eliminate the spacer region thus eliminating the autoprocessing step. The Codex® Acylase Panel (Codexis) contains PGAvariants that lack the spacer region (See e.g., US Pat. Appln. Publn.2010/0143968 A1). The PGA-encoding genes were cloned into the expressionvector pCK110900 (See, FIG. 3 of US Pat. Appln. Publn. No. 2006/0195947)operatively linked to the lac promoter under control of the laclrepressor. The expression vector also contains the P15a origin ofreplication and the chloramphenicol resistance gene. The resultingplasmids were transformed into E. coli W3110, using standard methodsknown in the art. The transformants were isolated by subjecting thecells to chloramphenicol selection, as known in the art (See e.g., U.S.Pat. No. 8,383,346 and WO2010/144103).

Example 2 Preparation of HTP PGA-Containing Wet Cell Pellets

E. coli cells containing recombinant PGA-encoding genes from monoclonalcolonies were inoculated into 180 μl LB containing 1% glucose and 30μg/mL chloramphenicol in the wells of 96 well shallow well microtiterplates. The cultures were grown overnight at 30° C., 200 rpm and 85%humidity. Then, 10 μl of each of the cell cultures were transferred intothe wells of 96 well deep well plates containing 390 mL TB and 30 μg/mLCAM. The deep-well plates were incubated for 3 hours (OD600 0.6-0.8) at30° C., 250 rpm and 85% humidity. The cell cultures were then induced byIPTG to a final concentration of 1 mM and incubated overnight under thesame conditions as originally used. The cells were then pelleted usingcentrifugation at 4000 rpm for 10 min. The supernatants were discardedand the pellets frozen at −80° C. prior to lysis.

Example 3 Preparation and Analysis of HTP PGA-Containing Cell Lysates

First, 250 μl lysis buffer containing 20 mM Tris-HCl buffer, pH 7.5, 1mg/mL lysozyme, and 0.5 mg/mL PMBS was added to the cell paste in eachwell produced as described in Example 2. The cells were lysed at roomtemperature for 2 hours with shaking on a bench top shaker. The platewas then centrifuged for 15 min at 4000 rpm and 4° C. The clearsupernatants used in biocatalytic reactions to determine their activitylevels.

The activity of the PGA variants was evaluated based on the efficiencyof the variant in removing the three phenyl acetate groups chemicallyattached to the A1 (glycine), B1 (phenylalanine), and B29 (Lysine)residues of insulin. HTP reactions were carried out in 96 well deep wellplates. First, 0.3 ml of the reaction mixture contained 0.1M Tris-HCl,pH 8.0, 5 g/L tri-protected insulin and HTP lysate between 25-125 μl(depending on the linear curve). The HTP plates were incubated inthermotrons (3 mm throw, model # AJ185, Infors at 30° C., 300 rpm, for 6or 22 hrs. The reactions were quenched with 300 μl acetonitrile andmixed for 3 mins using a bench top shaker. The plates were thencentrifuged at 4000 rpm for 2 mins and loaded into an HPLC for analysis.An Agilent eclipse XDB C18, 5 μm, 2.1×150 mm column was used to analyzethe HTP samples. The flow rate was set to 0.5 ml/min and the temperaturewas set to 50° C. Mobile phase A was water+0.05% TFA, and mobile phase Bwas acetonitrile+0.05% TFA. The runtime was 7.2 minutes, with injectionoverlap enabled. The gradient was 75% mobile phase A in 0.2 minutes, 55%mobile phase A in 4.9 minutes, 5% mobile phase A in 5.4 minutes, back to75% mobile phase A in 5.9 minutes.

Example 4 Preparation and Analysis of Lyophilized Lysates from ShakeFlask (SF) Cultures

Selected HTP cultures grown as described above were plated onto LB agarplates with 1% glucose and 30 μg/ml CAM and grown overnight at 37° C. Asingle colony from each culture was transferred to 50 ml of LB with 1%glucose and 30 ng/ml CAM. The cultures were grown for 18 h at 30° C.,250 rpm, and subcultured approximately 1:10 into 250 ml of TB containing30 μg/ml CAM, to a final OD₆₀₀ of 0.2. The cultures were grown for 135minutes at 30° C., 250 rpm, to an OD₆₀₀ between 0.6-0.8 and induced with1 mM IPTG. The cultures were then grown for 20 h at 30° C., 250 rpm. Thecultures were centrifuged 4000 rpm×20 min. The supernatant wasdiscarded, and the pellets were resuspended in 30 ml of 50 mM sodiumphosphate, pH 7.5. The cells were pelleted (4000 rpm×20 mM) and frozenat −80° C. for 120 minutes. Frozen pellets were resuspended in 30 ml of50 mM sodium phosphate pH 7.5, and lysed using a Microfluidizer system(Microfluidics) at 18,000 psi. The lysates were pelleted (10,000 rpm×60min) and the supernatants were frozen and lyophilized to generate shakeflake (SF) enzymes.

The activity of selected shake flask PGA variants was evaluated based onthe efficiency of the variants in removing the three phenyl acetategroups chemically attached to the A1 (glycine), B1 (phenylalanine), andB29 (lysine) residues of insulin. The shake flask reactions were carriedout in 96 well deep well plates. First, 0.3 ml of the reaction mixturecontained 0.1M Tris-HCl, pH 8.0, 5 g/L tri-protected insulin, shakeflask lysate between 0.1-0.8 g/L. The deep well reaction plates wereincubated in thermotrons (3 mm throw, model # AJ185, Infors) at 30° C.,300 rpm, for 22 or 6 hrs (22 hrs for round 1 evolution and 6 hrs forround 2 evolution). The reactions were quenched with 300 μl acetonitrileand mixed for 3 mins on a bench top shaker. The plates were thencentrifuged at 4000 rpm for 2 mins and loaded into HPLC for analysis. AnAgilent eclipse XDB C18, 5 μm, 2.1×150 mm columns was used to analyzethe HTP samples. The flow rate was set to 0.6 ml/min and the temperaturewas set to 50° C. Mobile phase A was water+0.05% TFA and mobile phase Bwas acetonitrile+0.05% TFA. The runtime was 18.2 minutes with injectionoverlap enabled. The gradient was 80% mobile phase A in 0-1 minutes, 60%mobile phase A in 12 minutes, 5% mobile phase A in 15 minutes, back to80% mobile phase A in 16 minutes.

Example 5 Round 1 Evolution Backbone Selection, Construction andScreening

Following the HTP protocol described in the above Examples, the Codex®Acylase Panel (Codexis) was evaluated using the shake flask protocolsdescribed in Example 4. One of the variants, designated as “Variant 1”from the Codex® Acylase Panel (SEQ ID NO:4) generated 54% free insulinwith 0.8 g/L shake flask lysate loading in 22 hrs. A substrateinhibition study was conducted using Variant 1 (See, FIG. 1). With theincreasing concentration of the tri-protected insulin substrate,generation of free insulin catalyzed by Variant 1 decreasedsignificantly. In 5 hrs., free insulin generation dropped from 82% with1 g/L substrate loading to 2% with 10 g/L substrate loading at a fixedenzyme loading of 0.8 g/L. While it is not intended that the presentinvention be limited to any particular mechanism, these results suggestthat higher concentrations of the tri-protected insulin substrate causedsubstrate inhibition. The amount of free insulin production increased atlower substrate concentration (See e.g., Wang et al., Biopolymer25:S109-S114 [1986]). Thus, one of the advantages provided by thepresent invention is the production of PGA variants that overcomesubstrate inhibition. This Variant was chosen as the backbone for thefirst round evolution. A homology model of Variant 1 was built using E.coli PGA as template (the E. coli PGA has 87% sequence identity to thewild-type K. citrophila PGA). Tri-protected insulin was docked into theactive site of Variant 1 to assess its interaction with PGA. Then, 96positions in its amino acid sequence were chosen for the first round ofevolution, covering the 1st layer (amino acid residues within 5-6 Å) andpart of the 2nd layer (amino acid residues within 6-12 Å) of the activesite and tri-protected insulin binding site. Two combinatorial librarieswere also designed, based on an analysis of the PGA panel screeningresults and consensus mutations. All of the HTP screenings for thevariants obtained in this first round of evolution were conducted usingthe same protocols as described above, with the final reaction timepoint being 22 hrs. Active mutations with corresponding fold-improvementin total activity over Variant 1 are shown in Table 5.1, below. In thisTable, the positive control is Variant 1 (SEQ ID NO:4).

TABLE 5-1 Active Mutations and FIOPC Results for Round 1 VariantsVariant # FIOPC Active Mutations (as compared to Variant 1) 2 +++ V56I;V264A; A308T; T379A; D484N 3 − G71F; D74G 4 + Q547K; L711Q; S750G 5 −G71F; D74G 6 +++ V264A; D484N; Q547K 7 − G71F; D74G 8 − G71F; D74G 9 ++V56I; A308T; T379A; D484N 10 − F367S; L754P 11 + V56I 12 − G71F; D74G 13++ D484N 14 +++ V264A; A308T; T379A; D484N; Q547K 15 + L754P 16 − G71F;D74G 17 − V264A; A308T; L711Q 18 − G71F; D74G 19 +++ V264A; T379A;D484N; S750G; L754P 20 + T379A; Q547K 21 ++ Q547K 22 ++ V56I; T379A;S750G 23 − Q547K; L711Q 24 − G71F; D74G 25 − Q547K; L711Q 26 − G71F;D74G 27 ++ V56I; D484N; L711Q 28 + Q547K; L754P 29 − G71F; D74G 30 +Y27F; T131N; G368D; S619K 31 − G71F; D74G 32 + Y27F; V28N; G368D; S372L;A616D; V618I; S619K 33 − G71F; D74G 34 + V28N; T131N 35 + Y27F; G368D;S372L; S619K 36 + Y27F; S372L; V618I; S619K 37 + Y27F; V56L; I127V;S372L; S619K 38 + Y27F; V28N; Q134H; A616D; S619K 39 ++ I127V; Q134H;G368D; V618I; S619K 40 + Y27F; I127V; T131N; A616D; V618I; S619K 41 +T131N; S372L 42 − G71F; D74G 43 + V56L; I127V; T131N; Q134H; G368D;A616D 44 − G71F; D74G 45 − G71F; D74G 46 ++ Y27F; V56L; G368D; S372L47 + V28N; I127V; T131N; G368D; S372L 48 ++ V28N; G368D; A616D; V618I;S619K 49 + V28N; V56L; S372L 50 + V28N; A616D; S619K 51 + Q134H 52 ++Y27F; V28N; T131N; Q134H; G368D; A616D; V618I; S619K 53 ++ Y27F; V28N;V56L 54 − G71F; D74G 55 + V56L; Q134H; G368D; A616D 56 − G71F; D74G 57++ Y27F; V28N; T131N; G368D; S619K 58 − G71F; D74G 59 ++ Y27F; S619K 60− G71F; D74G 61 ++ Y27F; V28N; V56L; T131N; Q134H; S619K 62 + Y27F;G368D; S372L; V618I; S619K 63 − G71F; D74G 64 + V56L; G368D; A616D;V618I 65 + Y27F; V28N; I127V; T131N; G368D; A616D; V618I; S619K 66 +V28N; T131N; S372L 67 +++ V28N; S619K 68 + V56L; S372L; A616D; S619K 69+++ Y27F; V28N; V56L; I127V; Q134H; G368D; S372L; S619K 70 ++ V28N;T131N 71 +++ Y27F; V56L; I127V; V618I; S619K 72 + I127V; T131N 73 −G71F; D74G 74 ++ Y27F; V28N; A616D; V618I; S619K 75 ++ V56L 76 ++ Y27F;V28N; V56L; G368D 77 − G71F; D74G 78 + Y27F; V56L; S372L 79 ++ S619K 80− G71F; D74G 81 + Y27F; V28N; I127V; Q134H; A616D; S619K 82 + V56L;I127V; T131N; S325R; V618I; S619K 83 + Y27F; V56L; I127V; Q134H; G368D;S372L 84 + Y27F; I127V; T131N; G368D 85 ++ Y27F; I127V; G368D; S372L;V618I; S619K 86 + V28N 87 + Y27F; V56L; I127V; Q134H; S372L; V618I;S619K 88 +++ Y27F; V28N; V56L; V618I 89 − G71F; D74G 90 + G368D; S372L;V618I; S619K 91 − G71F; D74G 92 ++ D74P 93 ++ G71A; D74S 94 ++ G71A;D74V 95 ++ G71A; D74V 96 + D74A 97 +++ G71A; D74V 98 ++ D74S 99 ++ D74T100 +++ G71A; D74N 101 +++ D74S 102 ++ D74G 103 ++ D74G 104 +++ D74N 105++ G71A; D74A 106 ++ D74V 107 +++ G71A; D74S 108 +++ D74N 109 ++ D74G110 +++ D74S 111 ++ D74P 112 ++ D74N 113 ++ D74N 114 + P49H; M697R 115 +D74H 116 + T32D 117 + P418Q 118 + V56I; D74L 119 +++ D381F 120 +++ K369C121 +++ N388G 122 +++ D381K 123 ++ A255G 124 ++ L253S 125 ++ Y27T; 126++ D381W 127 ++ N348R 128 ++ F254W 129 ++ D381W 130 ++ A373L 131 ++L257N 132 ++ L253V 133 ++ W370I 134 ++ A255L 135 ++ A373L 136 ++ T384R137 ++ Q380R 138 ++ D381M 139 ++ T384P 140 ++ D381I 141 ++ W240F 142 ++D381Q 143 ++ L387I 144 ++ D381G 145 ++ L253W 146 ++ A255M 147 ++ V360A148 ++ W370F 149 ++ N348H 150 ++ L253W 151 ++ N388S 152 ++ D381C 153 ++A255R 154 ++ L253F 155 ++ N348R 156 ++ D381L 157 ++ A255V 158 ++ A255Y159 ++ A132R 160 ++ A255L 161 + F454Y 162 + T129W 163 + N348H 164 +L387Q 165 + D381M 166 + L387F 167 + L387M 168 + S372A 169 + D381C 170 +T379S 171 + D381P 172 + L387G 173 + L387S 174 + N348D 175 + T378Q 176 +T379S 177 + I389P 178 + N348S 179 + T129W 180 + Q380K 181 + T379C 182 +A373L 183 + T384G 184 + T379R 185 + T384H 186 + D381R 187 + D381Q 188 +A132R 189 + L253T 190 + N348E 191 + T129K 192 + D381Y 193 + D381I 194 +A255L 195 + A373Y 196 + T133N 197 + V126I 198 + A365M 199 + D381C 200 +N348K 201 + T378C 202 + Y27H 203 + A373L 204 + D130E 205 + L387C 206 +A456T 207 + H156R 208 + T131R 209 + T384C 210 + D381R 211 + T453R 212 +T133C 213 + A255V 214 + Y27G 215 + Q380I 216 + L387E 217 + A255S 218 +T379L 219 + T133G; L557S 220 + D381V 221 + L387T 222 + T133G 223 + T133R224 + V391N 225 + Q134S 226 + T384N 227 + T131D 228 + D381C 229 + A373L230 + T384F 231 + T133S 232 + L387H 233 + F256Y 234 + T133S 235 + L387E236 + T384A 237 + A132T 238 + T379G 239 + W370V 240 + L387F 241 + F116I242 + T384R; F596L 243 + V391P 244 + T133A 245 + Y27V 246 + A160S 247 +L387E 248 + N348S 249 + L257R 250 + A373Q 251 + T133Q 252 + W26F 253 +Q380C 254 + A255F “−”: FIOPC < 0.7 “+”: FIOPC = 0.7 to 1.3 “++”: FIOPC =1.4 to 2.0 “+++”: FIOPC ≧ 2.1

Based on these results, Variants 6, 19, 14, 67, 88, and 53 were scaledup to shake flask volumes and their activities were analyzed using theprotocols described in the previous Examples. The results are shown inFIG. 2. Variant 6 generated 93% free insulin in 22 hrs at 0.8 g/L enzymeloading (See, FIG. 2) and achieved a better expression level incomparison with Variant 1. Variant 53 had a similar expression level asVariant 1, but generated 73% free insulin compared to 54% generated byVariant 1. Based on the results, Variant 6 (SEQ ID NO:6) was selected asa starting backbone for the next round (i.e., round 2) of evolution.This variant was also designated as an “expression hit.” Variant 53 (SEQID NO:8) was also selected as an alternative round 2 backbone and wasdesignated as an “activity hit.”

Example 6 Round 2 Library Construction and Screening

The most beneficial mutations identified from round 1 evolution wereD484N, V264A, Q547K, V56I, S750G, V56L, S619K, V28N, V618I, and T131N.Two combinatorial libraries were designed, based on analysis of thefirst round results, using both Variant 6 and Variant 53 as backbones.All of the HTP screening methods used for the round 2 variants wereconducted using the previously described protocols, with the finalreaction time point being 6 hrs. Active mutations with correspondingfold improvement in total activity over Variant 6 and Variant 53 areshown in Tables 6.1 and 6.2, below. Table 6.1 provides the results forvariants based on Variant 6, while Table 6.2 provides the results forvariants based on Variant 53.

TABLE 6.1 Active Mutations and FIOPC Results for Round 2 Variants Basedon SEQ ID NO: 6 (Variant 6) Variant # FIOPC Active Mutations (ascompared to Variant 6) 255 +++ V28N; G71A; D74V; S619K 256 +++ V28N;G71A; D74V; S619K 257 +++ G71A; D74N; S619K 258 +++ V28N; V56L; G71A;D74S; S619K 259 ++ G71A; D74V; V618I; S619K 260 ++ G71A; D74S; S619K 261++ V28N; V56L; G71A; D74V 262 ++ V28N; G71A; D74N; S619K 263 ++ G71A;D74V; S619K 264 ++ G71A; D74V 265 ++ G71A; D74V 266 ++ G71A; D74S 267 ++S619K 268 ++ V28N; S619K 269 ++ V28N; S619K 270 ++ V28N; S619K 271 ++V28N; S619K 272 ++ V28N; G71A; D74S 273 ++ S619K 274 ++ D74V; S619K 275++ G71A; S619K 276 ++ S619K 211 ++ V28N; S619K 278 ++ S619K 279 ++ S619K280 ++ S619K 281 ++ S619K 282 ++ S619K 283 + V28N 284 + V28N 285 +V618I; S619K 286 + D74V 287 + S619K 288 + S619K 289 + S619K 290 + G71A291 − D74T; K139I; S619K 292 − R455W “−”: FIOPC < 0.7 “++”: FIOPC = 0.7to 1.3 “++”: FIOPC = 1.4 to 2.0 “+++”: FIOPC ≧ 2.1

TABLE 6.2 Active Mutations and FIOPC Results for Round 2 Variants Basedon SEQ ID NO: 8 (Variant 53) Variant # FIOPC Active Mutations (ascompared to Variant 53) 293 +++ G71A; D74V; D484N; Q547K 294 +++ G71A;D74S; D484N; Q547K 295 +++ D484N; Q547K; S619K 296 +++ G71A; D74S;D484N; Q547K 297 +++ D74N; D484N 298 +++ G71A; D484N; Q547K; V618I;S619K 299 +++ D74N; V264A; D484N; Q547K 300 +++ D74T; D484N 301 ++ G71A;V264A; D484N 302 ++ V264A; D484N; Q547K 303 ++ V264A; D484N 304 ++ L56I;G71A; D74S; V264A; D484N; Q547K; S619K 305 ++ D74S; D484N; V618I; S619K306 ++ D484N 307 ++ D484N; Q547K 308 ++ G71A; D74N; D484N; V618I 309 ++D484N; Q547K 310 ++ F27Y; N28V; D484N; Q547K; V618I 311 ++ D484N; S619K312 ++ D484N; Q547K; V618I; S619K 313 ++ D484N; V618I 314 ++ D74N; Q547K315 ++ Q547K; V618I; S619K 316 ++ D74N; V264A 317 ++ Q547K; S619K 318 ++G71A; D74V; V264A; Q547K; V618I 319 + Q547K; S619K 320 + V264A; V618I;S619K 321 + Q547K; V618I 322 + D74V; Q547K; S619K; A741T 323 + S619K324 + V264A; Q547K 325 + Q547K; V618I; S619K 326 + Q547K 327 + D74V328 + D74T 329 + V618I; S619K 330 + V264A; S619K 331 + G71A; V264A;Q547K 332 + V618I 333 − V264A “−”: FIOPC < 0.7 “++”: FIOPC = 0.7 to 1.3“++”: FIOPC = 1.4 to 2.0 “+++”: FIOPC ≧ 2.1

Variants 6 (SEQ ID NO:6), 258 (SEQ ID NO:12) and 261 (SEQ ID NO:10) werescaled up in shake flasks and their activities analyzed using theprotocols described in the previous Examples. The results are shown inFIG. 3. As indicated, Variant 258 generated >99% free insulin in 6 hrsin <0.8 g/L shake flask lysate loading, thereby completely releasingsubstrate inhibition, whereas Variant 261 generated ˜90% free insulin in6 hrs at 0.8 g/L lysate loading.

Thus, the present invention provides PGA variants having an 8× foldimprovement in total activity. It was also determined that the S619Ksubstitution largely impacts activity whereas the D484N substitutionpredominantly impacts expression. S619K is located in the 1^(st) layerof insulin binding site and D484N is located in the 2^(nd) layer of theactive site.

Example 7 DMSO Tolerance

A DMSO tolerance study was conducted on the improved PGA variants(Variant Nos. 6, 258, and 261). The reactions were carried out inpresence of 0-50% v/v DMSO following the protocol described in Example4. The results (See, FIG. 4) indicate that all the variants tested lostactivity upon addition of DMSO to the test reactions. For example, at30% v/v DMSO, only 30% free insulin was generated by Variant 258.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. An engineered penicillin G acylase capable of removing the A1/B1/B29 tri-phenyl acetate protecting groups from insulin to produce free insulin, wherein said penicillin G acylase is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:2, 4, 6, 8, 10, and/or
 12. 2. The engineered penicillin G acylase of claim 1, wherein said penicillin G acylase comprises at least one mutation as provided in Table 5.1, Table 6.2, and/or Table 6.3.
 3. The engineered penicillin G acylase of claim 1, wherein said penicillin G acylase comprises SEQ ID NO:4, 6, 8, 10, or
 12. 4. The engineered penicillin G acylase of claim 1, wherein said penicillin G acylase is encoded by a polynucleotide sequence selected from SEQ ID NOS:3, 5, 7, 9, and
 11. 5. A vector comprising the polynucleotide sequence of claim
 4. 6. A host cell comprising the vector of claim
 5. 7. A method for producing free insulin, comprising: i) providing the engineered penicillin G acylase of claim 1, and insulin comprising A1/B1/B29 tri-phenyl acetate protecting groups; and ii) exposing said engineered penicillin G acylase to said insulin comprising A1/B1/B29 tri-phenyl acetate protecting groups, under conditions such that said engineered penicillin G acylase removes the A1/B1/B29 tri-phenyl acetate protecting groups and free insulin is produced.
 8. The method of claim 7, wherein said engineered penicillin G acylase produces more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more free insulin.
 9. The method of claim 7, wherein said penicillin G acylase comprises SEQ ID NO:4, 6, 8, 10, or
 12. 10. A composition comprising free insulin produced according to the method of claim
 7. 